Considering the importance of carotenoids in the human diet, their enhancement is a key trait in current breeding programs. This study assessed lutein, zeaxanthin, α-carotene, and β-carotene levels in the flesh of mature fruits from 257 global C. pepo accessions. Lutein and β-carotene were the most prevalent, with top accessions identified for each carotenoid. A panel of 120 accessions with reliable carotenoid contents and genetic diversity was analyzed using 23,111 GBS-generated SNPs in genome-wide association studies (GWAS). Three genomic regions (qtl1, qtl3, and qtl13) on chromosomes 1, 3, and 13 were significantly linked to carotenoid levels, with alternative alleles increasing the carotenoid content, leading to yellowish–orange flesh. Seven candidate genes were identified: CpTIC56, CpHSHP70, and CpPDL8, which regulate carotenoid biosynthesis in chloroplasts; CpSPX and CpPHO1, associated with phosphate homeostasis and carotenoid buildup; CpMYB106, co-expressed with carotenoid biosynthesis genes; and a CpPPR RNA-binding protein. RNA-seq data from yellow- and white-fleshed fruits supported their involvement in carotenoid accumulation. These results improve our understanding of the genetic control of carotenoid buildup in C. pepo fruit, supporting breeding efforts for improved nutritional quality.