The red imported fire ant (Solenopsis invicta) is a non-native invasive species that rapidly spread northward in the United States after its introduction from South America to the southern coast in the 1930s. Researchers predicted that the northward spread of this invasive ant would be limited by minimum temperatures with increased latitude and elevation in the Southern Appalachian Mountains. The presence of S. invicta at relatively high elevations north of their projected limits suggests greater cold tolerance than previously predicted; however, these populations might be ephemeral indications of strong dispersal abilities. In this study we investigated potential physiological and behavioral adaptations of S. invicta that would indicate acclimation to high elevation environments. We hypothesized that if S. invicta colonies can persist in colder climates than where they originated, we would find gradients in S. invicta worker cold tolerance along a montane elevational gradient. We also predicted that higher elevation S. invicta ants might require greater physiological costs to persist in the colder climate, so we measured colony lipid content to assess health status. Finally, we predicted that the ants might compensate for colder climates by adjusting microscale colony locations (e.g., nearer to radiant heat masses). For comparison, we also collected physiological temperature tolerance data for the co-occurring dominant native woodland ant Aphaenogaster picea. We found that S. invicta occurring at higher elevations exhibited greater physiological tolerance for cold temperatures as compared to lower-elevation conspecifics – a cold tolerance pattern that paralleled of the native A. picea ants along the same gradient. Whereas S. invicta had a higher tolerance for heat compared to A. picea, both exhibited a similar downward shift in thermal tolerances when moving up the elevational gradient. There was no change in S. invicta colony lipid content with elevation, suggesting that greater metabolic rates were not needed to sustain these ants through high-elevation winter dormancy. In addition, S. invicta ants at high elevations did not appear to offset the colder high-elevation temperatures by selecting nest sites proximate to thermal masses.