Improving the powdery mildew resistance of bitter gourd is highly important for achieving high yield and high quality. To better understand the genetic basis of powdery mildew resistance in bitter gourd, this study analyzed 300 lines of recombinant inbred lines (RILs) formed by hybridizing the powdery mildew-resistant material MC18 and the powdery mildew-susceptible material MC402. A high-density genetic map of 1222.04 cM was constructed via incorporating 1,996,505 SNPs generated by resequencing data from 180 lines, and quantitative trait locus (QTL) positioning was performed using phenotypic data at different inoculation stages. A total of seven QTLs related to powdery mildew resistance were identified on four chromosomes, among which qPm-3-1 was detected multiple times and at multiple stages after inoculation. By selecting 18 KASP markers that were evenly distributed throughout the region, 250 lines and parents were genotyped, and the interval was narrowed to 207.22 kb, which explained 13.91% of the phenotypic variation. Through RNA-seq analysis of the parents, 11,868 differentially expressed genes (DEGs) were screened. By combining genetic analysis, gene coexpression, and sequence comparison analysis of extreme materials, two candidate genes controlling powdery mildew resistance in bitter gourd were identified (evm.TU.chr3.2934 (C3H) and evm.TU.chr3.2946 (F-box-LRR)). These results represent a step forward in understanding the genetic regulatory network of powdery mildew resistance in bitter gourd and lay a molecular foundation for the genetic improvement in powdery mildew resistance.