The Tustumena Lake drainage in southcentral Alaska is glacially turbid and geologically young (<2,000 years old). Previous field studies identified at least three subpopulations of sockeye salmon Oncorhynchus nerka at Tustumena Lake, based on the distribution and timing of spawners. The subpopulations included early‐run salmon that spawned in six clearwater tributaries of the lake (mid August), lake shoreline spawners (late August), and late‐run fish that spawned in the lakeˈs outlet, the Kasilof River (late September). Our objective was to determine the degree of genetic differentiation among these subpopulations based on restriction enzyme analyses of the cytochrome b gene of mitochondrial DNA and analyses of four polymorphic allozyme loci. Mitochondrial DNA haplotype frequencies for outlet‐spawning sockeye salmon differed significantly from those of all other subpopulations. The most common (36%) haplotype in the outlet subpopulation did not occur elsewhere, thus suggesting little or no gene flow between outlet spawners and other spatially close subpopulations at Tustumena Lake. Allele frequencies at two allozyme loci also indicated a degree of differentiation of the outlet subpopulation from the shoreline and tributary subpopulations. Allele frequencies for three tributary subpopulations were temporally stable over approximately 20 years (based on a comparison to previously published results) despite initiation of a hatchery program in two of the tributaries during the intervening period. Collectively, our results are consistent with the hypothesis that significant genetic differentiation has occurred within the Tustumena Lake drainage since deglaciation approximately 2,000 years ago.