We used quinacrine hydrochloride and chromomycin A3staining techniques to examine chromosome-banding polymorphisms in lake troutSalvelinus namaycushfrom seven wild populations and two hatchery stocks from the Great Lakes region. Although considerable variation in the number of bands occurred among individuals examined by both staining techniques, banding patterns were constant for all cells of a given individual. The populations were differentiated into three groups on the basis of the number of chromomycin A₃ bands: a group from the northern shores of Lakes Superior and Huron and the inland lakes north of Lake Superior; a group from the southern shore of Lake Superior; and a group that comprised one wild population and one hatchery stock from Wyoming that were both originally transplanted from a Lake Michigan stock. Each population within these three groups had a unique distribution of chromomycin A3bands on specific chromosomes. Results for a given population were consistent when sampled for two consecutive years. The populations were very similar in the frequency of Q-band (quinacrine-stained) variants except for one polymorphism found in the Michipicoten Island, Ontario, population and another polymorphism, which was rare in the Seneca Lake, New York, population. The cytogenetic results were compared with allozyme results from the same populations. The populations were similarly differentiated by both the allozyme and cytogenetic data.