Genetic diversity analysis of worldwide Cucurbita genetic resources preserved in the Japanese Genebank can provide valuable information for breeding. In this study, 612 Cucurbita accessions of six species, including 40 accessions with no identification information, were genotyped with 30 SSR markers; 378 alleles were detected (12.6 alleles per marker; range, 4-24). By cluster analysis, the 40 unidentified and 53 likely misidentified accessions were (re)identified. The identification was verified by cluster analysis based on the sequence of the mitochondrial atp4-ccmC region. After correction of the identification information, C. pepo accessions had the highest diversity indices among the species analyzed and thus showed potential as an ideal genetic resource for breeding. Among the three major species (C. moschata, C. maxima, and C. pepo), the diversity indices of accessions from Japan were lower than those of overseas accessions, indicating that the overseas accessions preserved in Japan are more genetically diverse and can be used as materials for the development of new cultivars. STRUCTURE and principal coordinate analyses of C. moschata revealed that several Japan accessions constituted an indispensable part of global crop genetic resources owing to their differences from overseas accessions. Commercial cultivars of C. maxima showed genetic similarity to each other in principal coordinate analysis, suggesting that they may have similar breeding properties. This study corrects some identification errors in the Genebank and could help improve the breeding of Cucurbita vegetables.