Intra-cultivar selection, reselection from existing cultivars by using intra-cultivar variation, is an important method in plant breeding. The main objective of this study is to evaluate the relationship of 22 soybean cultivars and 21 parents by use of 48 simple sequence repeat (SSR) loci. The genetic identity of 3 cultivars, developed by introducing foreign genome DNA through the pollen tube, and their parents was 35.42%–95.83%. Cultivars derived from the same parent showed differences in identity because of the difference in exogenous DNA. The identity of 19 soybean cultivars breeded by intra-cultivar selection and their parents was 27.08%–89.58%. Six cultivars showed < 30% identity with their parents. The 43 cultivars were clustered into Northeast spring, Huanghuai and South groups; 8 cultivars could not be grouped with their corresponding parents. The variation was from natural mutation but may also come from natural outcrossing. Agronomic and molecular data revealed great differences, which shows that use of only agronomic data is limited in evaluating relationship of cultivars. The preserved loci between parents and selected cultivars showed 14 SSR loci distributed in 11 linkage groups, specific allele was observed more than 7 times at each loci, and 7 alleles preserved in different materials were related to yield and disease-resistance quantitative trait loci. The result suggested that selecting for improved agronomic traits of soybean from elite cultivars preserved some alleles related to important characters such as yield.