The hybrid breeding in soybean（Glycine max）mainly relies on the three-lines system derived from cytoplasmic male sterility. Identification of optimal restorer lines is therefore of importance. Since checking the fertility of F1 hybrids derived from the tested restorer lines crossing with sterile lines is time-consumption and laborious，genetic identification of the strong restorer-of-fertility（Rf）gene（s）and its use for markerassisted selection（MAS）will greatly improve the efficiency of breeding. In this study，the F2 segregation population derived from the CMS-RN type sterile line JLCMS204A（female parent）and the restorer line JLR230（male parent，containing the unknown Rf gene）was investigated. Through examining pollen fertility the Rf gene in the restorer line was controlled by a pair of dominant single gene. Based on the bulked segregant analysis（BSA）of both parents and two pools（fertile and semi-sterile），the genetic locus named GmRf1 was allocated on chromosome 16 flanked by simple sequence repeat（SSR）markers BARCSOYSSR_16_1069 and BARCSOYSSR_16_1076. By taking use of enzyme digestion amplified polymorphic sequences（dCAPS） makers，insertion deletion（InDel）makers and sequence tag site（STS）makers，GmRf1 was finally delimited between the marker dCAPS-1 and BARCSOYSSR_16_1076，in which the genetic distance were 0.1 cM and 0.3 cM， respectively. On the basis of the ZH13 v2.0 reference genome，GmRf1 was located between 32 708 896 bp and 32 932 950 bp with a physical distance of 224.1 kb. This study will lay the foundation for molecular marker assisted breeding of the restorer lines containing GmRf1 locus and the isolation of the GmRf1 gene.