Gasterosteus aculeatus (threespine stickleback) is a well-known model for behavioral and evolutionary studies. Rapid evolutionary radiations in postglacial timeframes have promoted distinct local populations with remarkable variation in biological characteristics. This study examines genetic and morphological variation among populations from the Thlewiaza watershed, specifically an isolated freshwater population in Nueltin Lake. Statistically significant genetic differences were observed using 11 microsatellite loci; FST values ranged from 0.29 (within watershed) to 0.48 (between watersheds) in comparison with the Nueltin Lake population. Gene flow between populations was likely inhibited due to isostatic rebound following the recent deglaciation of North America, 8.5 kya. In comparison with similar freshwater populations, the retention of defensive structures in G. aculeatus from Nueltin Lake was unexpected, and may reflect strong piscivorous predation pressures. Levels of differentiation, both genetic and morphological, observed in the Nueltin Lake population are highly significant, and should be recognised as a Designatable Unit (DU).