Drosophila, like most insects, are susceptible to low temperatures, and will succumb to temperatures above the freezing point of their hemolymph. For these insects, cold exposure causes a loss of extracellular ion and water homeostasis, leading to chill injury and eventually death. Chill-tolerant species are characterized by lower hemolymph [Na(+)] than chill-susceptible species and this lowered hemolymph [Na(+)] is suggested to improve ion and water homeostasis during cold exposure. It has therefore also been hypothesized that hemolymph Na(+) is replaced by other 'cryoprotective' osmolytes in cold-tolerant species. Here, we compared the hemolymph metabolite profiles of five drosophilid species with marked differences in chill tolerance. All species were examined under 'normal' thermal conditions (i.e. 20°C) and following cold exposure (4 h at 0°C). Under benign conditions, total hemolymph osmolality was similar among all species despite chill-tolerant species having lower hemolymph [Na(+)]. Using NMR spectroscopy, we found that chill-tolerant species instead have higher levels of sugars and free amino acids in their hemolymph, including classical 'cryoprotectants' such as trehalose and proline. In addition, we found that chill-tolerant species maintain a relatively stable hemolymph osmolality and metabolite profile when exposed to cold stress while sensitive species suffer from large increases in osmolality and massive changes in their metabolic profiles during a cold stress. We suggest that the larger contribution of classical cryoprotectants in chill-tolerant Drosophila plays a non-colligative role for cold tolerance that contributes to osmotic and ion homeostasis during cold exposure and, in addition, we discuss how these comparative differences may represent an evolutionary pathway toward more extreme cold tolerance of insects.