The purpose of this study was to investigate the possibilities of using objective methods of DNA analysis to verify the declared origin of reproductive material of Norway spruce in terms of the Czech Republic. Monitoring of the identity of reproductive material was carried out during three years, i.e., from seed collection to transplanted plants production. Reference samples from the 32 sets of reproductive material were obtained, analyses of microsatellite markers were performed, and the genetic compositions of sets were compared after statistical processing. Altogether, DNA analyses were performed on 1920 samples of plant material from 8 selected sources of forest reproductive material (units of forest reproductive material). Seven optimally polymorphic markers with sufficient informative value were selected for the subsequent evaluation of the genetic structure of the monitored sets of Norway spruce reproductive material by Bayesian clustering. Using the performed Structure analysis, the obtained profiles of 8 monitored units of reproductive material (4 sample sets from one units) of different origin were distinguishable from each other. Thus, these methodological procedures could be used in the state control systems to certify the origin of forest reproductive material and increase consumer protection of forest owners and nursery production in the Czech Republic. Identity of forest tree reproductive material is essential in artificial forest regeneration. A certain proof of origin is important for tracing back forest reproductive material. The Czech Republic as a member state of the European Union has the obligation to create a functioning control system for determination of forest reproductive material. This commitment is also based on international legislation (Council Directive 1999/105/EC on the marketing of forest reproductive material on the market). Its aim is to ensure clear identification of reproductive material from the acquisition to delivery to the consumer. The Directive 1999/105/EC is transposed into national legislation by Act No. 149/2003 Coll., on the marketing of forest reproductive material of forestry importance and artificial hybrids, intended for forest regeneration and afforestation, and amending certain related acts (Act on Trade in reproductive material of forest trees), as amended, the provisions of which include appropriate adjustment control measures. The existing legal regulations on forest reproductive material in the Czech Republic provide only the inspection of the master certificates and delivery papers as a control measure. The aim of the work was to verify the level of genetic diversity, heterozygosity and other genetic characteristics of selected samples of Norway spruce reproductive material based on analyses of microsatellite (SSR) markers, to determine genetic similarity of monitored sources of forest reproductive material (units of forest reproductive material), and to verify suitability of methodology for monitoring identity of Norway spruce reproductive material. DNA analyses were performed on 1920 samples of plant material from 8 selected sources of reproductive material. Sampling of reference samples was performed from the sets and units of reproductive material listed in Table 1. Seed collections for units of reproductive material was done from 35–60 trees from 8 recognized stands. Sampling of material to analyses was carried out over 3 years. From each of 8 reproductive material units there were analysed 60 samples from raw cones seeds, 60 samples from seeds after extraction and cleaning, 60 samples from seedling production and 60 samples from transplanted plant. The genomic DNA was extracted by the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer´s instruction. Seven polymorphic markers (PAAC23, SpAG2, WS00111.K13, WS00716.F13, WS0022.B15, WS0073.H08, WS0023.B03) with the corresponding predictive value were selected from the original set of 26 tested microsatellite markers. PCR and fragment analysis procedures were optimized for selected markers. PCR products were separated by capillary electrophoresis using the Applied Biosystems 3500 genetic analyser. The statistical programs CERVUS (Kalinowski et al. 2007), GenAlEx 6.503 (Peakall, Smouse 2006, 2012), the Bayesian clustering method implemented in the software STRUCTURE 2.3.4 (Pritchard et al. 2000; Falush et al. 2003, 2007; Hubisz et al. 2009) were used to analyse the genetic data. PCR products of the selected markers generated simple patterns and provided variable expected sizes of alleles. The genetic diversity parameters with the primer sequences of the studied markers are reported in Table 2. The most polymorphic locus was WS0023.B03 and the least was WS0073.H08. Genetic diversity characteristics of the 32 Norway spruce sample sets are given in Table 3. Nei’s genetic distances among 32 Norway spruce sample sets ranged from 0.04 to 0.325, and they are presented in Fig. 1, constructed on the basis of principal coordinate analysis (PCoA). The results of the AMOVA showed that variation among individuals was 10% and among the observed sample sets it was 3%. Pairwise population FST values ranging from 0.003 to 0.038 indicated low genetic differentiation between observed sets of sample. The structuring of investigated Norway spruce sample sets was also confirmed by various proportions of genetic profiles according to the Bayesian clustering method results (Fig. 2). Using the performed Structure analysis, the obtained profiles of 8 monitored units of reproductive material (4 sample sets from one units) of different origin were distinguishable from each other. These methodological procedures could be used in state control systems of forest reproductive material origin, and in order to increase consumer protection of forest owners and nursery production in the Czech Republic. [ABSTRACT FROM AUTHOR]