Few taxa exhibit the variability of sex-determining modes as amphibians. However, due to the presence of homomorphic sex chromosomes in many species, this phenomenon has been difficult to study. The Australian frog, Litoria aurea, has been relatively well studied over the past 20 years due to widespread declines largely attributable to chytrid fungus. However, it has been subject to few molecular studies and its mode of sex determination remained unknown. We applied DArTseq™ to develop sex-linked single nucleotide polymorphisms (SNPs) and restriction fragment presence/absence (PA) markers in 44 phenotypically sexed L. aurea individuals from the Molonglo River in NSW, Australia. We conclusively identified a male heterogametic (XX-XY) sex determination mode in this species, identifying 11 perfectly sex-linked SNP and six strongly sex-linked PA markers. We identified a further 47 moderately sex-linked SNP loci, likely serving as evidence indicative of XY recombination. Furthermore, within these 47 loci, a group of nine males were found to have a feminised Y chromosome that significantly differed to all other males. We postulate ancestral sex-reversal as a means for the evolution of this now pseudoautosomal region on the Y chromosome. Our findings present new evidence for the ‘fountain of youth’ hypothesis for the retention of homomorphic sex chromosomes in amphibians and describe a novel approach for the study of sex chromosome evolution in amphibia.