Tropical reptiles colonizing temperate regions face distinct physiological challenges due to thermal stress. Their long-term survival hinges on physiological acclimatization or adaptation to these novel thermal environments. Previous research on cold tolerance in introduced high-latitude Hemidactylus frenatus revealed that rural populations on Okinawajima Island, Japan exhibited significantly lower critical thermal minimum (CTmin) compared to their urban counterparts. However, whether this variation stems from acclimation or genetically fixed physiological differences remains unclear. To address this issue, we re-examined the cold tolerance of rural and urban populations after subjecting them to 2-week acclimation periods under high and low temperatures. Our experiments revealed that urban geckos exposed to the high-temperature treatment exhibited the highest CTmin, while rural geckos in the low-temperature treatment displayed the lowest CTmin. Urban geckos in the low-temperature treatment and rural geckos in the high-temperature treatment displayed intermediate CTmin values. These findings suggest that both acclimation and genetic differences contribute to the observed variations in cold tolerance among H. frenatus populations. The urban and rural populations are only 10 km apart, highlighting that evolutionary responses to thermal stress can occur rapidly over surprisingly small geographical scales in this species.