Among vertebrates, sex is determined by environmental or genotypic sex determination, such that sex is determined by incubation environment or by the genetic contribution of parents, respectively. The selective advantage of one mechanism over the other is unclear and our understanding of the evolution of environmental sex determination (ESD) is obscured by the lack of description of the mechanism's genetic architecture. In this study, a reptile with environmental sex determination, the leopard gecko, Eublepharis macularius, was tested for a genotype × environment interaction (G×E) with respect to sex-determining response to incubation temperature. Five sires were each mated to five unique dams. The eggs from each sire/dam combination were randomly assigned to one of three incubation temperatures and resultant hatchling sex ratios were measured. Temperature and temperature × dam (sire) interaction significantly affected treatment group sex. We report a statistically significant effect of genotype by environment interaction (G×E) on the offspring sex in E. macularius. G×E for ESD may provide an opportunity for evolution for different threshold conditions for sex determination in this ESD species.