Brown adipose tissue (BAT), present in many placental mammals, provides adaptive nonshivering thermogenesis (NST) for body temperature regulation and has facilitated survival in diverse thermal niches on our planet. Intriguingly, several key details on the molecular mechanisms of NST and their potential ecophysiological adaptations are still unknown. Comparative studies at the whole animal level are unpragmatic, due to the diversity and complexity of thermoregulation among different species. We propose that the molecular evolution of mitochondrial uncoupling protein 1 (UCP1), a central component for BAT thermogenesis, represents a powerful opportunity to unravel key questions of mammalian thermoregulation. Comparative analysis of UCP1 may elucidate how its thermogenic function arose, how environmental selection has shaped protein function to support ecophysiological requirements, and how the enigmatic molecular mechanism of proton leak is governed. Several approaches for the assessment of UCP1 function in vitro have been introduced over the years. For comparative characterization of UCP1, we put forward the overexpression of UCP1 orthologues and mutated variants in a mammalian cell system as a primary strategy and discuss advantageous aspects in contrast to other experimental systems. In turn, we suggest how remaining experimental caveats can be solved by complimentary test systems before physiological consolidation in the animal model. Furthermore, we highlight the appropriate bioenergetic techniques to perform the functional analyses on UCP1. The comparative characterizations of diverse UCP1 variants may enable key insights into open questions surrounding the molecular basis of NST.