Maize stalk rot is a major disease prevalent across major corn production regions in China, posing a significant threat to both yield and grain quality. Therefore, the identification of elite germplasm and underlying gene resources for stalk rot resistance is of great significance. In this study, through large-scale phenotyping of germplasm, we identified an elite maize inbred line, CNH3323, which showed high resistance to maize stalk rot. Using it as the resistant parent, we developed two segregating populations, one comprising 280 F_2:3 families and another comprising 410 doubled haploid (DH) lines. The phenotypic evaluations for stalk rot resistance were conducted for two segregating populations under both natural infection and artificial inoculation conditions across two locations over two years. Based on the genotyping of segregating populations and the construction of linkage maps, QTL mapping for maize stalk rot resistance was performed. The results showed that a total of 24 QTLs associated with stalk rot resistance were detected across different environments, distributed on eight chromosomes except for chromosomes 5 and 8. Notably, an environment stable QTL with major-effect, designated as qRfg3.03, was consistently mapped to the bin3.03 region on chromosome 3 using different populations, which explained up to 30.36% of the phenotypic variance. This study provides important candidate loci for the genetic dissection of maize stalk rot resistance and also offers valuable gene resources for maize improvement and the breeding for the resistant to stalk rot, which holds significant theoretical research and practical application value.