Turtles demonstrate variability in sex determination and, hence, constitute an excellent model for the evolution of sex chromosomes. Notably, the sex determination of the freshwater turtles from the family Chelidae, a species-rich group with wide geographical distribution in the southern hemisphere, is still poorly explored. Here we documented the presence of an XX/XY sex determination system in seven species of the Australasian chelid genera Chelodina, Emydura, and Elseya by conventional (karyogram reconstruction, C-banding) and molecular cytogenetic methods (comparative genome hybridization, in situ hybridization with probes specific for GATA microsatellite motif, the rDNA loci, and the telomeric repeats). The sex chromosomes are microchromosomes in all examined species of the genus Chelodina. In contrast, the sex chromosomes are the 4th largest pair of macrochromosomes in the genera Emydura and Elseya. Their X chromosomes are submetacentric, while their Y chromosomes are metacentric. The chelid Y chromosomes contain a substantial male-specific genomic region with an accumulation of the GATA microsatellite motif, and occasionally, of the rDNA loci and telomeric repeats. Despite morphological differences between sex chromosomes, we conclude that male heterogamety was likely already present in the common ancestor of Chelodina, Emydura and Elseya in the Mesozoic period.