Cucurbita moschata, commonly known as pumpkin, is highly valued for its nutritional content, stress tolerance, fruit phenotypic diversity, excellent characteristics of easy-planting and transportation, and long shelf-life, which makes it recommended as an essential crop for the mitigation of food and nutritional challenges. However, the absence of a high-quality reference genome hinders genetic improvement studies on pumpkins. Here, we successfully assembled the telomere-to-telomere (T2T) genome of C. moschata L. (2n = 40) with a total size of 289.6 Mb (Contig N50 = 11.2 Mb). It comprises all 40 telomeres, 8 gaps, and 29 901 protein-coding genes (BUSCO = 97.9%), and half of the chromosomes are composed of a single contig. Additionally, to investigate the genetic basis of fruit traits in pumpkin, we developed an F₂ genetic population consisting of 200 individuals and constructed a high-density genetic linkage map using the assembled genome as the reference. Quantitative trait locus (QTL) mapping of 20 fruit traits was applied. An AUX/IAA gene and a β-tubulin gene were identified as candidate genes regulating longitudinal flesh thickness and transverse/longitudinal diameter ratio, respectively. In summary, the genome assembly for C. moschata and QTL mapping results provide valuable resources for molecular breeding and functional genomics studies in the pumpkin.

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