Recent research in monotremes has revealed independent origins of two sex chromosome systems in mammals. The therian XY system evolved after the divergence of the monotremes and established the Y-localised Sry gene as the therian master sex determining (MSD) gene, required for male sexual development. The multiple sex chromosomes of monotremes (platypus and echidna) share no homology to the therian sex chromosomes, lack the Sry gene and evolved from different autosomes. The identity of the MSD gene of monotremes has remained a mystery. The discovery of the Y-localised copy of the evolutionarily conserved sex differentiation gene, Amh (Anti-Mullerian hormone), provides a candidate MSD gene in monotremes. Y-localised Amh genes have independently evolved as the MSD gene in multiple vertebrate species. Characterisation of the monotreme X- and Y-localised Amh gametologues shows substantial divergence between the X- and Y-copies of the gene, protein and promoter region. These differences may underlie sex specific expression patterns and functions for the Amhx and Amhy gametologues. We further show that Amhx is expressed in the ovary and testis of adult monotremes and in the bipotential gonad, while Amhy is exclusively expressed in males, with expression seen in the adult testis and in the male bipotential gonad prior to and during, sexual differentiation, consistent with a role in sexual determination.