Environmental temperature strongly influences the adaptation dynamics of amphibians, whose limited regulation capabilities render them susceptible to thermal oscillations. A central element of the adaptive strategies is the transcription factors (TFs), which act as master regulators that orchestrate stress responses, enabling species to navigate the fluctuations of their environment skillfully. Our study delves into the intricate relationship between TF expression and thermal adaptation mechanisms in the Rhinella spinulosa populations. We sought to elucidate the dynamic modulations of TF expression in prometamorphic and metamorphic tadpoles that inhabit two thermally contrasting environments (Catarpe and El Tatio Geyser, Chile) and which were exposed to two thermal treatments (25 °C vs. 20 °C). Our findings unravel an intriguing dichotomy in response strategies between these populations. First, results evidence the expression of 1374 transcription factors. Regarding the temperature shift, the Catarpe tadpoles show a multifaceted approach by up-regulating crucial TFs, including fosB, atf7, and the androgen receptor. These dynamic regulatory responses likely underpin the population’s ability to navigate thermal fluctuations effectively. In stark contrast, the El Tatio tadpoles exhibit a more targeted response, primarily up-regulating foxc1. This differential expression suggests a distinct focus on specific TFs to mitigate the effects of temperature variations. Our study contributes to understanding the molecular mechanisms governing thermal adaptation responses and highlights the resilience and adaptability of amphibians in the face of ever-changing environmental conditions.