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Distinctness, uniformity and stability (DUS) testing of cultivars through morphological descriptors is an important and compulsory part of soybean breeding. Molecular markers are usually more effective and accurate in describing the genetic features for the identification and purity assessment of cultivars. In the present study, we assessed the distinctness and uniformity of five soybean cultivars using both single nucleotide polymorphism (SNP) markers developed by specific-locus amplified fragment sequencing (SLAF-seq) technology, and simple sequence repeat (SSR) markers. The phylogenetic tree and principal component analysis (PCA) from both the SLAF-seq and SSR methods showed a clear distinction among cultivars Zhonghuang 18, Zhonghuang 68 and Zhonghuang 35, while no clear distinction was observed between cultivars Zhonghuang 13 and Hedou 13. Using the SLAF-seq method, we determined the proportion of homozygous loci for the five soybean cultivars. The heterozygosity of each individual plant was estimated for the assessment of cultivar purity and the purity levels of the five soybean cultivars ranged from 91.89% to 93.96%. To further validate the applicability of the SLAF-seq approach for distinctness testing, we used the SNP information of 150 soybean cultivars with different origins. The cultivars were also distinguished clearly. Taken together, SLAF-seq can be used as an accurate and reliable method in the assessment of the distinctness and uniformity of soybean cultivars. [ABSTRACT FROM AUTHOR]