We studied the differentiation of North American squash bee (Apidae: Apiformes) populations by using geometric wing morphometry to evaluate hypotheses for the origin and possible expansion route of bees after the dispersal of their Cucurbita floral hosts. Two hypotheses consider the possible impact of early human cultures on the distribution of this mutualistic species complex: 1) the geographic range of the bees naturally expanded after the hypsithermal range expansion of the wild buffalo gourd, Cucurbita foetidissima Kunth; or 2) the bees shifted to mesophytic gourds of the C. pepo group at the Mexican Gulf coast and tracked the cultivation and/or trade by early human hunter-gatherer societies in the context of fishing technology along stream valleys to northeastern America. We morphometrically measured 228 specimens of Peponapis pruinosa (Say) collected at 15 sites between Mississippi and Ontario, Canada. Twenty-three landmarks were taken from the right forewings. The resulting measurements were analyzed by multivariate statistics. Our results support the first hypothesis of a western origin of North American squash bee populations and a mainly natural range expansion during the hypsithermal range expansion of the buffalo gourd.