Pleistocene glaciers profoundly shaped the distribution and evolutionary history of many subterranean organisms in the northeast United States such as Stygobromus allegheniensis. During the last glacial maximum, many of these organisms were subject to sub-zero conditions associated with the Laurentide Ice Sheet. Such conditions would explain why S. allegheniensis from the Ice Caves of Sam’s Point area of New York are able to survive being encapsulated by ice. The Ice Caves at Sam’s Point experience seasonal freezing and prolonged ice formation during winter, whereas Clarksville Cave is warmer and maintains thermally stable liquid water year-round. To determine whether freeze tolerance is a retained ancestral trait in S. allegheniensis or a locally derived adaptation, we compared cold tolerance in amphipods from these two cave systems. Amphipods from Clarksville Cave showed some tolerance to near-freezing temperatures and partial ice contact, despite such conditions never occurring in their habitat, indicating probable retention of a vestigial physiological trait. However, they were highly susceptible to complete ice encasement, in stark contrast to Ice Cave specimens, which tolerate prolonged freezing conditions. Survival differed significantly between the two populations under complete ice encasement. Our results suggest that ancestral cold-tolerance mechanisms have persisted in S. allegheniensis since the Pleistocene, with populations from caves that naturally freeze nowadays showing further enhancement of freeze resistance through local adaptation.