Pumpkin seed (Cucurbita pepo L.) is a nutritionally valuable food and a significant source of income globally. Pumpkin seeds are rich in oil, protein, unsaturated fatty acids and tocopherols, which are associated with improved human health. Understanding the genetic diversity among pumpkin accessions varying in seed nutrition traits is necessary for designing sound breeding strategies for developing superior cultivars. In the current study, 26 simple sequence repeats (SSR) markers were used to assess genetic relatedness among 29 C. pepo accessions varying in seed oil, seed protein, seed-coat phenotype, seed size and fatty acid composition. The SSR markers revealed 102 alleles averaging 3.92 alleles per loci and mean polymorphic information content (PIC) of 0.44. Eleven of the markers had a PIC of ≥0.5. Ward dendrogram and principle component analysis based on seed traits grouped the genotypes into two major clusters corresponding to subspecies pepo and texana, with all the reduced-hull accessions grouping within the former. Collectively, this data suggests wide phenotypic (seed traits) and genotypic variation within C. pepo that may be exploited to develop superior reduced-hull cultivars.