Maize rough dwarf disease (MRDD), caused by Fijivirus, poses a significant threat to global maize production.  Using a recombinant inbred line (RIL) population derived from the resistant parent CML199 and the susceptible parent Zheng58, we identified three MRDD resistance QTLs on chromosomes 2, 6, and 9, accounting for 12.71, 5.89, and 11.04% of the total phenotypic variation, respectively.  Among them, the major locus qMrdd3 on chromosome 2 demonstrated incomplete dominance, conferring a resistance enhancement of 26.36–34.47% across diverse environments.  Fine-mapping refined qMrdd3 to a 227.7-kb interval containing five candidate genes, among which Zm00001d002441 was specifically upregulated in the resistant near-isogenic line (NIL-R) following RBSDV infection.  Additionally, two co-segregating markers were developed to facilitate efficient marker-assisted selection.  Introgression of qMrdd3 into Zheng58 and Chang7-2 enhanced field resistance by 38.84 and 26.47%, respectively.  This study provides a valuable genetic resource for MRDD resistance breeding through QTL dissection, elite germplasm development, and marker-assisted breeding.