Interpretation of the genetic composition and taxonomic history of wolves in the western Great Lakes region (WGLR) of the United States has long been debated and has become more important to their conservation given the recent changes in their status under the Endangered Species Act. Currently, the two competing hypotheses on WGLR wolves are that they resulted from hybridization between (i) grey wolves (Canis lupus) and western coyotes (C. latrans) or (ii) between grey wolves and eastern wolves (C. lycaon). We performed a genetic analysis of sympatric wolves and coyotes from the region to assess the degree of reproductive isolation between them and to clarify the taxonomic status of WGLR wolves. Based on data from maternal, paternal and bi-parental genetic markers, we demonstrate a clear genetic distinction between sympatric wolves and coyotes and conclude that they are reproductively isolated and that wolf–coyote hybridization in the WGLR is uncommon. The data reject the hypothesis that wolves in the WGLR derive from hybridization between grey wolves and western coyotes, and we conclude that the extant WGLR wolf population is derived from hybridization between grey wolves and eastern wolves. Grey-eastern wolf hybrids (C. lupus × lycaon) comprise a substantial population that extends across Michigan, Wisconsin, Minnesota and western Ontario. These findings have important implications for the conservation and management of wolves in North America, specifically concerning the overestimation of grey wolf numbers in the United States and the need to address policies for hybrids.