The egg-laying mammals (monotremes) form the most basal, extant mammalian lineage and possess both mammalian and reptilian features. Most intriguingly, the monotremes (platypus and echidna) have a multiple XY sex chromosome system with 5X and 5Y chromosomes in the male platypus and 5X and 4Y in the echidna male. These multiple sex chromosomes are unique to this mammalian group, having evolved after divergence of the monotremes from therian mammals. Although monotremes possess genes common to the vertebrate sexual differentiation pathways, they lack the master sex determinant (MSD) gene, Sry required for testis development in therian mammals. The monotreme MSD is unknown. In a number of teleost fish, a Y chromosome specific Anti-Müllerian hormone (Amh) gene has been recruited independently to the role of MSD. Monotremes are the only mammals known to carry the Amh gene on sex chromosomes (bearing an X and Y copy). As such, the Y localised Amh gene (Amhy) is the primary candidate MSD in monotremes. Here we characterise the monotreme Amh genes showing that the Amhx and Amhy proteins contain the conserved features and predicted structures of other Amh proteins suggesting that they also function through Amh receptor II signalling. We demonstrate that the Amhy gene and protein of monotremes has significantly diverged from its X-localised gametologue. Differences between the Amhy and Amhx promoters and introns may underlie differential expression of the genes and support the posited role of Amhy as the monotreme MSD.