The evolution of sex determination remains one of the most fascinating enigmas in biology. Transitions between genotypic sex determination (GSD) and temperature-dependent sex determination (TSD) have occurred multiple times during vertebrate evolution, however, the molecular basis and consequences of these transitions in closely related taxa remain unresolved. Here I address a critical question: Do species with GSD derived from ancestors possessing TSD retain any ancestral thermal sensitivity in the developmental pathways underlying gonadal differentiation? Results from an expression study of a gene involved in early gonadogenesis in GSD (Apalone mutica) and TSD (Chrysemys picta) turtles, support the hypothesis that Wt1 in A. mutica displays such a relic thermal sensitivity. This retention is likely enabled by Sf1, a gene immediately downstream from Wt1 whose expression is independent of temperature in this species. My results constitute the first empirical evidence of a GSD vertebrate exhibiting thermal sensitivity in the expression of a gene regulating gonadogenesis. This novel finding reveals an undocumented source of raw material for future evolutionary change that may exist in other GSD taxa, and one that enhances the evolutionary potential of the gene networks underlying sexual differentiation and contributes to the astonishing ability of sex-determining mechanisms.