The stem rot caused by the soil-borne pathogenic fungus Sclerotium rolfsii Sacc. is a devastating disease that limits peanut production in China and other countries around the world. To explore novel resistant germplasm resources against this disease, our study conducted a seedling-stage resistance identification on 703 peanut germplasm resources. The results showed significant virulence differences among three pathogenic isolates, as well as certain resistance differences among the germplasm resources from various origins. Evaluation of seedling-stage identified three germplasm resources SNHD132, SNHD072, and Baihuashengfen exhibited resistance against all three isolates. Subsequently the three resistant varieties were subjected to both in vitro inoculation and field inoculation for resistance identification. The results indicated that after in vitro inoculation, the lesion expansion on the three resistant varieties was significantly smaller than that on the susceptible control materials. After field inoculation, the three resistant materials showed significantly lower plant mortality rate, seed infection rate, and yield loss compared to the susceptible control varieties. Combining seedling-stage, in vitro, and field identifications, this study obtained resistant resources and provides excellent materials and a theoretical basis for future breeding of new varieties resistant to stem rot disease and related genetic research.