Climate change is dramatically altering our planet, yet our understanding of mechanisms of thermal tolerance is limited in wild birds. We characterized natural variation in heat shock protein (HSP) gene expression among tissues and populations of free-living Tree Swallows (Tachycineta bicolor). We focused on HSPs because they prevent cellular damage and promote recovery from heat stress. We used quantitative PCR to measure gene expression of three HSPs, including those in the HSP70 and HSP90 families that have robust experimental connections to heat in past literature. First, to evaluate how tissues and, by extension, the functions that they mediate, may vary in their thermal protection, we compared HSP gene expression among neural and peripheral tissues. We hypothesized that tissues with particularly vital functions would be more protected from heat as indicated by higher HSP gene expression. We found that brain tissues had consistently higher HSP gene expression compared to the pectoral muscle. Next, we compared HSP gene expression across four distinct populations that span over 20 degrees of latitude (>2300 km). We hypothesized that the more southern populations would have higher HSP gene expression, suggesting greater tolerance of, or experience with, warmer local conditions. We observed largely higher HSP gene expression in more southern populations than northern populations, although this pattern was more striking at the extremes (southern Indiana vs. Alaska) and it was stronger in some brain areas than others (ventromedial telencephalon vs. hypothalamus). These results shed light on the potential mechanisms that may underlie thermal tolerance differences among populations or among tissues.