Microsatellite or Simple Sequence Repeats (SSR) marker is a new type of molecular marker developed recently, which can be used in genotype identification, pedigree analysis, and estimation of genetic distance. Using 5 pairs of SSR primer, 21 polymorphic fragments were obtained in 15 soybean germplasm. The number of alleles at each SSR locus is from 3 to 6, and the range of gene diversity is 0.439-0.668. In addition, analysis of genetic distance was also performed in this study. Pedigree analysis showed that mutation occurred for individuals in the F8 population of RIL after multigeneration meiosis, which was caused because of the changes in the number of repeat unit, to a limited extent.