Thermal tolerance is a key physiological trait that often varies among populations and species, and can be a key determinant of current and future geographic distributions. The cellular basis for organismal differences in thermal tolerance is an active area of research, with chemical constituents of the cytosol and cell membrane likely playing important roles in maintaining cell function at low temperatures. For freeze‐avoiding organisms, the abundance and makeup of sugars and related molecules depress the freezing point, reducing the risk of ice formation. At less extreme temperatures, remodeling of membrane lipids can help maintain fluidity (and function) at cold temperatures. We have found striking differences in critical thermal minima (CTmin) of worker bumble bees reared in common‐garden conditions from queens collected at low and high latitudes. We compared metabolomic and lipidomic signatures of these bees to identify key molecules that likely underlie differences in cold tolerance. PCA analyses of GC‐MS data for aqueous abdominal extracts revealed clear separation of low and high latitude populations and reveal higher levels of several key molecules, including L‐proline, in the high latitude population. Lipid extractions and subsequent GC‐MS reveals variation in fatty acid composition of nutrients (triacylglycerols) and of cellular membranes, for which phospholipid composition is also critical for function at low temperatures. This broad spectrum approach to metabolomics and lipidomics allows us to compare a wide range of metabolites between bees differing in cold tolerance, facilitating ongoing research on the mechanistic basis for this physiological trait linked to the ecology of diverse organisms.Support or Funding InformationThis material is based upon work supported by the National Science Foundation under Grant Nos. DEB‐1457659 and RII Track 2‐1826834. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.