Microsatellites are one of the most suitable markers for variety identification as it has great discrimination power for varieties with narrow genetic variation. The polymorphism level between forty microsatellite primer pairs and 148 soybean varieties was investigated through fluorescence based automatic detection system. A set of 16 primer pairs showed highly reproducible and polymorphic in these varieties. A total of 204 alleles were detected by using 16 microsatellite markers. The number of alleles per locus ranged from 6 to 28 with an average of 12.75 alleles per locus. The average polymorphism information content (PIC) was 0.86 ranging from 0.75 to 0.95. Two hundred four microsatellite loci were used to calculate Jaccard’s distance coefficients for unweighted pair group method using the arithmetic averages cluster analysis. These varieties were separated into several distinctive groups corresponding to varietal types. All of the varieties were perfectively discriminated by markers genotypes. This information may be useful to compare through genetic relationship analysis between existing and candidate varieties in distinctive tests and protection of plant breeders’ intellectual properties rights through variety identification.