Pumpkin (Cucurbita pepo), a versatile-member of the Cucurbitaceae-family, holds significant commercial-value in numerous countries, both as a cultivated-field-crop and a home-garden-staple. As with all crops, pumpkins are subject to diseases that can limit crop quality and yield. Considering the importance of the subject, in this research, SSR-markers were employed to investigate genetic-diversity and population-structure within a collection of 41 pumpkin-genotypes for the first time. At the same time, the screening for resistance to Phytophthora melonis, one of the important diseases of pumpkin globally, was carried out at greenhouse-conditions. In addition, defense-genes were analyzed against P. melonis, accordingly. The results showed that the pumpkin-genotypes have a high genetic-diversity. The 13-primers produced a total of 73-amplicons, of which 72 displayed polymorphic-patterns. Based on the dendrogram generated, the genotypes could be clustered into five distinct groups. Variance-analysis, showed that the related-genotypes had significant-reaction to the P. melonis. The genotypes were stratified into four groups based on their varying degrees of infection. (resistant, tolerant, sensitive and very sensitive) on the cut-off line found on the dendrogram. Comparison between the classification of genotypes in terms of disease-resistance and their grouping based on SSR-marker, there was a high agreement between the disease-severity and the dendrogram of the molecular-marker comprised their accordance with the disease-severity-percentage of the related pumpkin-genotyped accordingly. Moreover, the related defense-genes overexpressed following inoculation with to P. melonis, of which MYB, bHLH87 and ERF014 were more predominant significantly. The findings of this investigation elucidate the underlying mechanisms governing pumpkin resistance to Phytophthora root and crown rot. The data generated from this study can be leveraged to identify resistant pumpkin genotypes, promising SSR-marker and candidate genes for genetic enhancement of susceptible pumpkin cultivars and the subsequent development of P. melonis-resistant pumpkin varieties.