The prerequisite of genomic research such as genetic mapping is construction of a suitable segregating population and its reasonable evaluation before use. A recombinant inbred line (RILs), NJRIKY, was constructed through a cross between Kefeng 1 and Nannong 1138-2. The present study was focused on evaluation of genetic structure of the RILs through the simple sequence repeats (SSRs) analysis. A total number of 138 pairs of SSRs primers covering the whole soybean genome were randomly selected for polymorphic analysis between two parents. 86 pairs of which were polymorphic and polymorphic rate is up to 62.32%. 90 polymorphic loci were found. Further analysis with polymorphic SSRs loci on the sampled lines indicated that not only segregation of most SSRs loci conformed to the ratio of 1:1, but also almost each sampled line tended to homozyge. The distribution of genotypic composition was normal. The population is ideal for genetic mapping and other genomic research.