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Temperatures of inland aquatic habitats are increasing with climate change, and understanding how fishes respond physiologically to thermal stress will be crucial for identifying species most susceptible to these changes. Desert fishes may be particularly vulnerable to rising temperatures because many species occupy only a fraction of their historical range and occur in habitats with already high temperatures. Here, we examined endocrine and metabolic responses to elevated temperature in Amargosa pupfish, Cyprinodon nevadensis amargosae. We studied C. n. amargosae from two habitats with distinct thermal conditions: the Amargosa River, which experiences diurnally and seasonally variable temperatures (0.2-40 degrees C); and Tecopa Bore, a spring and marsh fed by hot groundwater (47.5 degrees C) from an artesian borehole. These allopatric populations differ in morphology, and prior evidence suggests that temperature might contribute to these differences via altered thyroid hormone (TH) regulation of morphological development. Here, we document variation in hepatic iodothyronine deiodinase type 2 (dio2) and type 3 (dio3) and TH receptor beta (tr beta) gene transcript abundance between the Amargosa River and Tecopa Bore wild populations. Fish from these populations acclimated to 24 or 34 degrees C retained differences in hepatic dio2, dio3 and tr beta mRNAs and also varied in transcripts encoding the TH membrane transporters monocarboxylate transporter 8 (mct8) and organic anion-transporting protein 1c1 (oatp1c1). Tecopa Bore pupfish also exhibited higher dio2 and tr beta mRNA levels in skeletal muscle relative to Amargosa River fish. Muscle citrate synthase activity was lower at 34 degrees C for both populations, whereas lactate dehydrogenase activity and lactate dehydrogenase A-chain (ldhA) transcripts were both higher and 3,5,3’-triiodothryonine responsive in Tecopa Bore pupfish only. These findings reveal that local population variation and thermal experience interact to shape how pupfish respond to elevated temperatures, and point to the need to consider such interactions in management actions for desert fishes under a changing climate.