Egg-laying mammals (monotremes) lack the therian sex determination gene SRY and their sexual development pathway has not been characterised. The candidate sex determining gene of monotremes is a Y-localised copy of the Anti-Müllerian Hormone gene. AMH has conserved roles in vertebrate sexual development and has become the sex-determining gene in a range of non-mammalian vertebrates. In this work, we characterise the monotreme X- and Y-localised AMH gametologues and provide new evidence supporting AMHY as the male sex determining gene of monotremes. The AMH gametologues of platypus and echidna are located in the oldest strata of the X and Y chromosomes however, Y chromosome degradation has altered the genomic context of the monotreme AMHY gene compared to AMHX and therian AMH genes. We show that the AMHX and AMHY genes have undergone significant divergence at the promoter, gene and protein level but have retained the conserved features of mammalian TGF-β signalling molecules. Access to echidnas during early developmental stages has allowed us to investigate gene expression during monotreme sexual determination. We show that AMHX is expressed in the developing gonad of both males and females during the period of sexual determination, while AMHY is expressed throughout these fetal stages only in the developing testis. Expression of AMHY in the developing male gonad during sex determination provides strong support for its role in male sex determination. Functional analysis was carried out in the chicken by ectopic expression of the monotreme AMHX or AMHY genes in the chicken embryo. In this system, chicken AMH masculinizes the female chicken gonad, however the monotreme proteins did not affect gonadal development, likely due to an inability of the proteins to signal through the chicken AMH receptor. In conclusion, our results provide additional evidence in support of AMHY as the male sex determining gene in monotremes.