Branched glycerol dialkyl glycerol tetraethers (brGDGTs) in lake sediments are increasingly being used to reconstruct past temperatures. However, recent studies suggest that brGDGT distributions and concentrations vary with lake size and environmental conditions, such as seasonality and its effects on water column temperature and chemistry. To test their use as a paleothermometer in high-altitude environments of mid-latitude North America, we analyzed brGDGT distributions in lake surface sediments across a range of lake depths and elevations in the Rocky Mountains of Wyoming and Colorado. Our results suggest that brGDGT distributions and the MBT′5Me index correlate with water column temperatures, which are sensitive to both lake water depth and air temperatures. Based on these relationships, we developed a calibration to mean summer air temperatures using a Bayesian regression model that incorporates the MBT′5Me index and lake water depth. We applied our calibration to lake sediments from Lower Paintrock Lake in northern Wyoming to test its use as a paleothermometer. Reconstructed temperature trends are consistent with pollen-inferred temperatures at the same site and with known regional climate history, demonstrating that our calibration can be successfully applied to infer temperatures in high-altitude environments of mid-latitude North America.