Production of summer squash, Cucurbita pepo, can be severely limited by viral pathogens and powdery mildew. Resistance has been introgressed from Cucurbita moschata, and resistant hybrids have been commercially deployed. Our objective was to assess genetic affinities of such hybrids with susceptible, open-pollinated cocozelle and zucchini cultivars, and two disease-resistant lines derived from six generations of backcrossing to a susceptible zucchini cultivar. Amplified fragment length polymorphism (AFLP) EcoRI/MseI primer combinations were employed and, based on the resulting polymorphic bands, genetic similarities were estimated, and an unweighted pair group method using arithmetic average (UPGMA) cluster analysis was conducted. The open-pollinated cocozelle cultivars clustered with the resistant hybrids. The hybrids had greater similarities with one another than did the open-pollinated cultivars. The zucchini cultivars and their resistant backcross lines formed their own exclusive cluster. However, the resistant backcross lines showed less than 0.80 similarity with their recurrent parent. As the chromosome number of Cucurbita is high (2n = 2x = 40) and the resistances are inherited monogenically and oligogenically, these results, after six generations of backcrossing, cannot be explained by classical genetic linkage.