Nascent sex chromosomes offer a unique opportunity to investigate the evolutionary fate of genes recently trapped in non-recombining segments. A house-keeping gene (MED15) was recently shown to lie on the nascent sex-chromosomes of the European tree frog (Hyla arborea), with different alleles fixed on the X and the Y chromosomes. Here we document a polymorphism (glutamine deletion) in the X copy of the gene, and use population surveys and experimental crosses to test whether this polymorphism is neutral or maintained by sex-antagonistic selection. Tadpoles from parents of known genotypes revealed significant discrepancies from Mendelian inheritance, suggesting possible sex-antagonistic effects under laboratory conditions. Quantitatively, however, these effects did not meet the conditions for polymorphism maintenance. Furthermore, field estimates of female genotypic frequencies did not differ from Hardy–Weinberg equilibrium and allelic frequencies on the X chromosome did not differ between sexes. In conclusion, although sex-antagonistic effects cannot be excluded given the laboratory conditions, the X-linked polymorphism under study appears neutral in the wild. Alternatively, sex-antagonistic selection might still account for the fixation of a male-specific allele on the Y chromosome.