Recent genetic data indicate that the eastern wolf is not a subspecies of the gray wolf (Canis lupus), but is a North American wolf more similar to the red wolf (C. rufus) and closely related to the coyote (C. latrans). The eastern wolf has been proposed as a separate species, C. lycaon. The largest protected area containing this wolf is Algonquin Provincial Park in Ontario, Canada, which is bounded to the south by areas containing the Tweed wolf or eastern coyote, a hybrid of the western coyote and eastern wolf. We assessed the relationships of animals in the park by using DNA profiles that comprised the genotype from 17 autosomal and 4 Y-linked microsatellite loci and the mitochondrial DNA control region. These profiles were used to establish maternity, paternity, and kin relationships for 102 wolves that were studied from 24 packs over a 12-year period. Genetic data do not support the hypothesis that a pack comprises an unrelated breeding pair and their offspring. There is evidence of frequent pack splitting, pack fusion, and adoption. Some unrelated individuals in the packs were identified as immigrants into the park. We found high levels of genetic structuring between the Tweed wolves to the southeast and the Algonquin Park wolves (R_ST = 0.114). Lower levels of genetic differentiation with animals to the north and west (R_ST = 0.057 and R_ST = 0.036) and high genetic diversity suggest that park animals are not an island population but the southern part of a larger metapopulation of C. lycaon.