Temperature variation is one of the primary challenges facing ectotherms, and the ability to tolerate a range of thermal environments is critical for setting current and future species distributions. Low temperature is particularly challenging for ectotherms because winter conditions have strong latitudinal and temporal variation. Lower lethal temperature (LLT) is a common metric of cold tolerance used in studies of local adaptation and plasticity. Comparisons of LLT across groups typically assume parallel S-shaped survival curves, but genetic variation in the shape of survival vs. temperature curves has not been assessed. Here, we measured the ability of 36 lines of the Drosophila Genetic Reference Panel (DGRP) to survive a 1-h cold shock at seven ecologically relevant low temperatures (-1 to -7 °C) to create a high-resolution response curve for each genotype. We observed surprising variation both in the magnitude of survival and in the shapes of the response curves, with the curves clustering into four distinct shapes. To encompass variation in the shapes of these survival curves, we developed a new cold tolerance metric, cumulative cold tolerance (CCT). By comparing our survival data with climatological data, we propose that variation in the shapes of cold-survival curves arose from weak selection pressure to survive intermediate subzero temperatures in this mid-latitude population of flies. Using publicly available genome sequence and transcript expression data for these lines, we identified several candidate genes associated with CCT, and using transgenic RNAi, we confirmed a functional role for many of these genes.