Abstract – Two modal size groups of sexually mature Arctic charr (Salvelinus alpinus) differing in shape and found at different depths in Lake Aigneau in the Canadian sub-Arctic are described and tested for genetic and ecological differentiation. Forms consisted of a small littoral resident, mean size 21.7 cm, and a large profundal resident, mean size 53.9 cm. Mitochondrial DNA analysis indicated that seven of eight haplotypes were diagnostic for either the littoral or profundal fish, with 66.6% of the variation being found within form groupings. Pairwise tests of microsatellite data indicated significant differences in nine of 12 loci and a significant difference between the forms across all tested loci. Molecular variation was partitioned to 84.1% within and 15.9% between forms and suggestive of either restricted interbreeding over time or different allopatric origins. Stable isotope signatures were also significantly different, with the profundal fish having higher δ13C and δ15N values than the littoral fish. Overlap and separation, respectively, in the range of form δ13C and δ15N signatures indicated that carbon was obtained from similar sources, but that forms fed at different trophic levels. Littoral fish relied on aquatic insects, predominantly chironomids. Profundal fish were largely piscivorous, including cannibalism. Predominantly empty stomachs and low per cent nitrogen muscle-tissue composition among profundal fish further indicated that the feeding activity was limited to the winter when ice-cover increases the density of available prey at depth. Results provide evidence of significant differences between the modal groups, with origins in both genetics and ecology.