Ultraviolet‐B radiation (UV‐B; 280–320 nm) penetrates some aquatic habitats to biologically significant depths and can alter life histories of aquatic organisms, including algae, zooplankton, fish, and amphibians. Although major species differences have been documented for UV‐B sensitivity, few studies have examined differences between populations of the same species. Previous work has suggested the hypothesis that larval long‐toed salamanders (Ambystoma macrodactylum) from valley populations (∼100 m elevation) were more sensitive to UV‐B exposure than larvae from mountain populations (above 500 m elevation). To test this hypothesis in the absence of possible other confounding environmental effects, we brought early‐stage embryos into the laboratory from three valley populations and five mountain populations from Oregon and Washington and raised them under identical conditions without UV‐B for two months after hatching. Larvae from each population were then placed under UV‐B lighting, and we recorded their growth and survivorship for three weeks. Larvae from all populations had higher mortality when exposed to UV‐B than when shielded from UV‐B. However, individuals from low‐elevation populations exposed to UV‐B had significantly lower survivorship than did those from high‐elevation populations, suggesting an elevational difference in UV‐B sensitivity. In all populations, individuals exposed to UV‐B were smaller than shielded individuals after one week. If UV‐B is a factor in determining the long‐term persistence of some amphibian species, an understanding of within‐species variation is critical.