Characterizing the molecular network underlying temperature-dependent (TSD) and genotypic (GSD) sex determination, including patterns across closely related taxa, is crucial to elucidate the still enigmatic evolution of sex determining mechanisms in vertebrates. Here we examined the expression of an important gene for sexual differentiation common to both systems, Sf1, at male- and female-producing temperatures, in TSD (Chrysemys picta) and GSD turtles (Apalone mutica). We tested the hypotheses that Sf1 expression responds to temperature consistently across TSD turtles but is unaffected in GSD turtles, and that this differential expression starts no earlier than the onset of the thermosensitive period (TSP). As expected, Sf1 expression was thermally insensitive in A. mutica (GSD). Although Sf1 exhibited a differential expression by temperature in C. picta, the expression pattern differed from other TSD turtles (Trachemys scripta), perhaps reflecting divergence of the gene regulatory networks underlying sex determination over evolutionary time. Most notably, Sf1 was differentially expressed in C. picta (significantly higher at the male-producing temperature) before the onset of the TSP, implying that in TSD taxa significant thermal effects may occur early in development. This result may reconcile field observations where temperatures experienced prior to the TSP have an effect on sex ratios, thus challenging traditional TSP models. Importantly, the molecular factors that render TSD mechanisms thermosensitive remain unknown, and potential candidates are genes that express differentially before the onset of the TSP (genes shaping or opening the TSP-window rather those acting once the TSP window has opened). Therefore, our findings make Sf1 one such potential candidate.