Seed coat color, one of the most important morphological markers, is changed from black to yellow during the artificial selection of cultivated soybean from wild soybean. Therefore, discovering genes related to the seed coat color is very important not only for evolutionary study but also for breeding practice in soybean. In this study, Jin1265-1 (yellow seed coat) and its derived line J1265-2 with brown seed coat were used to check whether they had the same genetic background using 161 SSR markers. Meanwhile, The coding sequence and promoter region of a candidate gene (GmF3'H) which was related to seed coat color in soybean was also amplified and sequenced. SSR marker analysis revealed that two lines with different seed coat color had similar genetic background and they were near isogenic lines. There were no sequence difference between two lines in coding region and 1465bp promoter region of GmF3'H , suggesting that gene controlling brown seed coat in J1265-2 was different from the known allele.