The breeding of hybrid soybean varieties is mainly based on the three-line method （based on cytoplasmic male sterility）. As the male parent of the hybrid， the restorer-of-fertility （Rf ） gene in the restorer line plays a decisive role. In the former study， we mapped the Rf gene GmRf1 for the soybean RN type cytoplasmic male sterile line. This study conducted functional annotation， subcellular location prediction， sequence alignment and differential expression analysis of candidate genes in the physical interval of GmRf1. We identified that the PPR （Pentatricopeptide repeats） gene Glyma.16G161900 encoding for 576 amino acids encoded in male restorer line JLR230 is GmRf1. By crossing Williams82 containing GmRf1 with the female sterile line JLCMS204A， the pollen fertility of F₁ plants was identified demonstrating that GmRf1 could restore the fertility of CMS-RN sterile lines. We further developed functional molecular markers Rf1-dCAPS-2 and Rf1-dCAPS-3 based on the single nucleotide mutation sites of GmRf1 between female and male parents. Collectively， this study laid a theoretical and technical foundation for the future marker-assisted selection of GmRf1， as well as the creation of new restorer lines through genetic engineering.