Distinguishing genetically differentiated populations within hybrid zones and determining the mechanisms by which introgression occurs are crucial for setting effective conservation policy. Extensive hybridization among grey wolves (Canis lupus), eastern wolves (C. lycaon) and coyotes (C. latrans) in eastern North America has blurred species distinctions, creating a Canis hybrid swarm. Using complementary genetic markers, we tested the hypotheses that eastern wolves have acted as a conduit of sex-biased gene flow between grey wolves and coyotes, and that eastern wolves in Algonquin Provincial Park (APP) have differentiated following a history of introgression. Mitochondrial, Y chromosome and autosomal microsatellite genetic data provided genotypes for 217 canids from three geographic regions in Ontario, Canada: northeastern Ontario, APP and southern Ontario. Coyote mitochondrial DNA (mtDNA) haplotypes were common across regions but coyote-specific Y chromosome haplotypes were absent; grey wolf mtDNA was absent from southern regions, whereas grey wolf Y chromosome haplotypes were present in all three regions. Genetic structuring analyses revealed three distinct clusters within a genetic cline, suggesting some gene flow among species. In APP, however, 78.4% of all breeders and 11 of 15 known breeding pairs had assignment probability of Q0.8 to the Algonquin cluster, and the proportion of eastern wolf Y chromosome haplotypes in APP breeding males was higher than expected from random mating within the park (P<0.02). The data indicate that Algonquin wolves remain genetically distinct despite providing a sex-biased genetic bridge between coyotes and grey wolves. We speculate that ongoing hybridization within the park is limited by pre-mating reproductive barriers.