Powdery mildew infection is a significant challenge in butternut squash (Cucurbita moschata) production during winter in Hainan, China. The tropical climate of Hainan promotes powdery mildew infection, resulting in substantial yield losses. By utilizing transcriptome and genome sequencing data, SNPs and potential genes associated with powdery mildew resistance in butternut squash were identified. The analysis of differentially expressed genes (DEGs) following powdery mildew infection revealed several genes involved in resistance, with particular focus on a resistance (R) gene cluster that may be linked to the observed resistance. Two MLO genes in clade V from Cucurbita moschata may not be directly associated with resistance in the two genotypes studied. These findings are expected to contribute to the development of genetic tools for improving powdery mildew resistance in Cucurbita crops, thereby reducing yield losses and enhancing the sustainability of butternut squash production in Hainan and other regions.