The North American spiny-cheek crayfish, Orconectes limosus (Rafinesque, 1817), a widespread invader in Europe, seems to have been introduced there successfully only once. According to available literature, 90 individuals of unclear origin were released in Poland in 1890. Despite this apparent bottleneck, the species has successfully colonized various aquatic habitats and has displaced native crayfish species in many places. To test whether different European populations were likely to have come from a single source and to identify their possible origin, we analyzed the diversity of the mitochondrial gene for cytochrome c oxidase subunit I (COI) of O. limosus individuals from Europe and from its original range in North America, including the presumed source region of European populations, the Delaware River watershed (eastern USA). Two haplotypes were found in European populations. One haplotype was widespread; the other was present in a single population. In contrast, 18 haplotypes were detected in North America. This result supports the hypothesis of a single overseas introduction of O. limosus and suggests that the high invasion success of this species was not limited by an introduction bottleneck. Two divergent clades were detected in North American O. limosus populations. One, which includes the dominant haplotype in Europe, was found in a large part of the species' present range. The 2^nd (diverging by >1%) was mostly restricted to a limited area in southeastern Pennsylvania. Orconectes limosus populations in the northern part of the species' North American range, at least some of which are nonindigenous themselves, may share the source area with European O. limosus. The endangered status of O. limosus populations in southeastern Pennsylvania and northeastern Maryland, where much of the species' genetic diversity resides, should be considered in conservation management.