Wolf packs generally consist of a breeding pair and their maturing offspring that help provision and protect pack young. Because the reproductive tenure in wolves often is short, reproductively mature offspring might replace their parents, resulting in sibling or parent-offspring matings. To determine the extent of incestuous pairings, we measure relatedness based on variability in 20 microsatellite loci of mated pairs, parent-offspring pairs and siblings in two populations of gray wolves. Our 16 sampled mated pairs had values of relatedness not overlapping those of known parent-offspring or sibling dyads, which is consistent with their being unrelated or distantly related. These results suggest that full siblings or a parent and their offspring rarely mate and that incest avoidance is an important constraint on gray wolf behavioral ecology.