The lake trout Salvelinus namaycush is the focus of an international effort by fishery management agencies to restore this once-native species to Lake Ontario. Evaluation of reproductive success and comparisons among stocked lake trout strains require genetic markers. We used allozyme variation to make genetic comparisons among strains of lake trout stocked into Lake Ontario. Forty-two proteins presumed to be encoded by 102 loci were resolved electrophoretically; 18 loci were polymorphic. Among 16 samples from five principal origins (Finger Lakes, Great Lakes basin, northern New York–Adirondack, Manitoba, and mixed origins–Lake Ontario), the average observed heterozygosity was 0.029, and the proportion of polymorphic loci was 0.125. Significant differences occurred among the 16 samples at all 18 possible locus comparisons. The average Fst value was 0.14. Hierarchical analysis indicated that the variation among samples within an origin was greatest within the Great Lakes basin, which included the greatest number of samples and represented the largest geographic area. Most variation observed among samples, however, occurred among origins. The 1983 and 1984 hatchery year classes produced from the 1978 Seneca brood stock did not differ significantly. The 1981 Seneca brood stock more closely resembled the sample of wild lake trout from Seneca Lake than progeny from the 1978 brood stock. The closer affinity of the 1981 brood stock to wild Seneca lake trout may be due to the larger number of wild adults from Seneca Lake used to found these fish than was used to establish the 1978 brood stock. Siscowet (“fat”) and “lean” lake trout from Lake Superior were significantly different from each other; however, the level of variation between them was not greater than that among samples from other origins, and thus did not support recognition of siscowet lake trout as a distinct subspecies. The Jenny Lake strain possessed a genetic affinity to the siscowet sample from Lake Superior. Historical reports about the origin of this strain suggest that siscowet lake trout from northern Lake Michigan may have been used to found this strain. Therefore, the Jenny Lake strain may serve as a gene source for the establishment of the siscowet lake trout in Lake Ontario. The substantial differentiation among lake trout strains reported here supports the feasibility of using allozyme markers to identify the parental sources of naturally produced young.