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• Nine Impatiens macrovexilla populations were selected from diverse natural habitats. • These populations all had specific floral traits valuable for breeding. • Seven iPBS primers were optimized to genotype individual accessions. • Genetic variation of 77 % was within populations, whilst 23 % was among populations. • Specific biotypes of I. macrovexilla could then be used for targeted breeding. Impatiens macrovexilla Y. L. Chen is a potential ornamental herbaceous plant originated from a relatively small area of subtropical Karst region in southern China, and natural populations of this species presented strong adaptability to a range of environmental conditions, with strong horticultural characteristics including bright colour, large flowers, early flowering, long flowering, and floriferousness. Overall, these adventive traits are significant for future selective breeding. Using a 'jumping gene' based or retrotransposon-based Inter-Primer Binding Site (iPBS) marker system to evaluate the genetic variation of nine I. macrovexilla populations collected from contrasting environments. The electrophoretic patterns of the PCR products produced for all the accessions were applied to evaluate the genetic diversity and differentiation between populations. The genotyping results distinguished all the 91 individual accessions in nine I. macrovexilla populations from varied microclimates, and population G was separated as a particular variety (I. macrovexilla var. yaoshannensis) which agrees with former botanical classifications about this species. Results from the AMOVA analysis showed that the greatest genetic variation (77 %) was observed within populations whilst 23 % was recorded among populations, indicating a wide range of biotypes at each location where germplasm collected. Results from our PCoA analysis, the phylogenetic dendrogram, and the Bayesian clustering analysis showed obvious subdivision among populations analyzed. The genetic relationships for population G (Jinxiu) and population H (Gantang) were the most different from each other and showed the highest genetic distance of 0.254. Characteristics of specific traits for each population and the genetic relationships of these populations could be utilized in optimizing potential parental candidate for cross combinations. Variations of the genetic pattern and the relationships among and within the populations will impact profoundly on our selective breeding of the species in the future. [ABSTRACT FROM AUTHOR]