One area of risk assessment for transgenic crops concerns cross-compatible wild and weedy relatives. In squash (Cucurbita pepo), free-living populations are diverse in their distributions, ecologies, histories, and genetic and phenotypic compositions. As part of the effort to understand this diversity, random amplified polymorphic DNA (RAPD) data were collected from 37 wild or weedy populations and 16 cultivars, which together represented all infraspecific taxa of C. pepo. Twenty-six primers yielded 70 scorable and variable markers. The presence/absence of bands for these markers produced a data matrix which was analyzed using cluster analysis. The analysis confirmed the relationships among infraspecific taxa that had been revealed, in part, in previous genetic analyses (e.g., isozymes, chloroplast DNA restriction-site mutations, inter simple sequence repeats). Also supported were findings of varying degrees of gene flow from cultivars into free-living populations. Some of the RAPD variation in subsp.ovifera var.ozarkana populations was found to be correlated with the distribution of the drainage systems along which these populations are dispersed. Finally, the RAPD results support the idea that transgenic gene flow experiments with free-living populations should consider using representatives from each of the three free-living taxa, as well as from genetically or ecologically distinct populations within these taxa.