Soybean cultivars with genetically low levels of stachyose enhance the utilization of soybean in food as well as feed uses. The objective of this research is to obtain the information on indirection selection of soybean lines with low stachyose content using DNA marker based on RS2 (rs2) gene. Two genetic populations were developed from the crosses of three parents (116-13 parent : low stachyose content, PI417227 and PI506903 parents: normal stachyose content). Twenty F2 plants of RS2_ genotype and twenty F2 plants of rs2rs2 genotype from each populations were harvested. Content of stachyose was detected by HPLC. Stachyose contents (g/kg) of 116-13, PI417227, PI506903 parents were 3.7, 23.7, and 17.8, respectively. In population 1, stachyose content 20 F2 plants with RS2_ genotype was 14.8 – 24.1 and stachyose content 20 F2 plants with rs2rs2 genotype was 2.1 – 4.7. In population 2, stachyose content 20 F2 plants with RS2_ genotype was 12.4 – 19.7 and stachyose content 20 F2 plants with rs2rs2 genotype was 2.1 – 5.0. Mean difference between RS2_ genotype and rs2rs2 genotype in population 1 and 2 was highly significant. From this results, selection of genetic lines with low stachyose content by DNA marker based on RS2 (rs2) gene will be possible.