Southern rust (Puccinia polysora Underw.) is a major disease that can cause severe yield losses in maize (Zea mays L.). In our previous study, a major gene RppP25 that confers resistance to southern rust was identified in inbred line P25. Here, we report the fine mapping and candidate gene analysis of RppP25 from the near-isogenic line F939, which harbors RppP25 in the genetic background of the susceptible inbred line F349. The inheritance of resistance to southern rust was investigated in the BC₁F₁ and BC₃F₁ populations, which were derived from a cross between F939 and F349 (as the recurrent parent). The 1:1 segregation ratio of resistance to susceptible plants in these two populations indicated that the resistance is controlled by a single dominant gene. Ten markers, including three simple sequence repeat (SSR) markers and seven insertion/deletion (InDel) markers, were developed in the RppP25 region. RppP25 was delimited to an interval between P091 and M271, with an estimated length of 40 kb based on the physical map of B73. In this region, a candidate gene was identified that was predicted to encode a putative nucleotide-binding site leucine-rich repeat (NBS-LRR) protein. Two co-segregated markers will aid in pyramiding diverse southern rust resistance alleles into elite materials, and thereby improve southern rust resistance worldwide.