Recombinant inbred lines (RIL), which were generated by single seed descend method (SSD) with multiple generations from the F2 population, are commonly used in studies of the genetic mapping and the quantitative trait locus (QTL) mapping. Tracking and analyzing the genetic differences in generations of RILs might be of significance to assist the RIL population construction. In this study, 18 SSR markers being polymorphic with parents were used to track the genetic diversity of F1 to F10 population during the RIL construction in sesame. With the increase of RIL population generations, the levels of polymorphism became decreased constantly from 87.54% of F2 to 3.33% of F10. The percentage of loci showing heterozygous decreased from 100% of F1 to 0.19% of F10, and the coefficient of genetic similarity increased from 0.6669 of F2 to 0.9908 of F10, and the genetic distance decreased from 0.2592 of F2 to 0.0094 of F10. The percentage of the polymorphism, the heterozygous loci, the genetic similarity coefficient as well as the genetic distance were variable in F2 to F6, while the level of variations became smaller in generations of F6 to F10. The clustering results showed that F2 plants were randomly classified, while in F3 to F10 generations the plants derived from the same line were clustered together. F5 to F7 populations are ideal to select residual heterozygous lines. The high-generation populations (>7 generations, i.e. F8) are qualified for the genetic mapping and QTL mapping. The genetic background of F8 population tends to be consistent, which provides theoretical reference for the determination of self-crossing generation in sesame hybridization breeding.