Southern rice black-streaked dwarf virus disease (SRBSDVD) is one of the most serious viral diseases. Exploration of resistant germplasm resource and identification of resistance-conferring gene become of interest in rice breeding. Here, by testing for SRBSDVD resistance in a breeding population by artificial inoculation under both field and greenhouse conditions followed by RT-PCR detection, individual plants showing a segregation on resistance and susceptibility were observed implying the presence of a new recessive virus resistance locus. We assembled the resistance and susceptibility bulks by pooling the resistant and susceptible plants, respectively. By genotyping using the whole genome breeding gene chip (GSR 40K) technology, this resistance gene against southern rice black-streaked dwarf virus disease was delimited in a lower resolution. Collectively, this study provided a resistance resource/gene valuable in resistance breeding against southern black streaked dwarf disease.