During development, sexual differentiation results in physiological, anatomical and metabolic differences that implicate not only the gonads but also other body structures. Sex in Leopard geckos is determined by egg incubation temperature. Based on the premise that the developmental decision of gender does not depend on a single gene, we performed an analysis on E. macularius to gain insights into the genes that may be involved in gonads’ sexual differentiation during the thermosensitive period. All the genes were identified as differentially expressed at stage 30 during the labile phase of sex differentiation. In this way, the expression of genes known to be involved in gonadal sexual differentiation, such as WNT4, SOX9, DMRT1, Erα, Erβ, GnRH, P450 aromatase, PRLand PRL-R, was investigated. Other genes putatively involved in sex differentiation were sought by differential display. Our findings indicate that embryo exposure to a sex-determining temperature induces differential expression of several genes that are involved not only in gonadal differentiation, but also in several biological pathways (ALDOC, FREM1, BBIP1, CA5A, NADH5, L1 non-LTR retrotransposons, PKM). Our data perfectly fit within the new studies conducted in developmental biology, which indicate that in the developing embryo, in addition to gonadal differentiation, sex-specific tissue and metabolic polarization take place in all organisms.