Empirical studies on the relative roles of occasional XY recombination versus sex-chromosome turnover in preventing sex-chromosome differentiation may shed light on the evolutionary forces acting on sex-determination systems. Signatures of XY recombination are difficult to distinguish from those of homologous transitions (i.e., transitions in sex-determination systems that keep sex-chromosome identity): both models predict X and Y alleles at sex-linked genes to cluster by species. However, the XY-recombination model specifically predicts the reverse pattern (clustering by gametologs) for those genes that are directly involved in sex determination. Hence, the latter model can only be validated by identification of an ancestral sex-determining region (SDR) with trans-species polymorphism associated to sex. Here we combine a candidate-gene approach with a genome scan to identify a small SDR shared by four species of a monophyletic clade of European tree frogs. This SDR encompasses at least the N-terminal part of Dmrt1 and immediate upstream sequences. Our findings provide definitive evidence that sex-chromosome homomorphy in this clade results only from XY recombination, and take an important step toward the identification of the sex-determining locus. Moreover, the sex-diagnostic markers we identify will enable research on environmental sex reversal in a wider range of frog species.