Understanding rapid adaptation to novel environments is essential as we face increasing climatic change. Invasive species are an ideal system for studying adaptation as they are typically introduced to novel environments where they must adapt if they are to persist. We used the invasive cane toad, Rhinella marina, to investigate the contribution of plasticity and evolution to rapid adaptation in a novel environment. Rhinella marina is a neotropical toad that has invaded areas with climates outside of its native environmental niche. The goal of this research was to understand how cane toads persist in northern Florida, the coldest region of their combined natural and invasive range, and originally thought to be beyond their thermal breadth. We measured Critical thermal minima in cane toads from the original, warm introduction location (Miami), and their northern range edge (Tampa) to determine whether northern toads were more cold-tolerant, and to examine the contribution of adaptive plasticity and evolution to any changes in tolerance. Our results show that following acclimation to cold temperatures, southern toads are less tolerant of cold than northern toads. This persistent population difference implies selection for cold-tolerance in northern populations. Differences in individual responses indicate that plasticity is also involved in this response. Our findings have implications for conservation because predatory cane toad invasions threaten local faunas, especially native amphibians. Characterizing specific adaptive mechanisms that allow R. marina to expand its range will identify evolutionary processes that shape a highly successful invasive species.