The current model of mammalian sex chromosome evolution postulates two independent sex chromosome origins from different autosomes in monotremes and therian mammals (marsupials and placentals) after their divergence 187 million years ago. In therian mammals, establishment of the sex determination gene SRY is assumed to be one of the early steps in sex chromosome evolution. In platypuses (which are monotremes) chromosome 6 shares extensive homology with the therian X chromosome, including the SOX3 gene from which SRY evolved. Therefore, platypus chromosome 6 provides unique insight into differentiation of therian XY. Cytological analysis of meiosis and sperm revealed that platypus chromosome 6 is segregated non-randomly at the first meiotic division in males, which results in a bias of chromosome 6 in X- and Y-bearing sperm. During meiosis we observed non-pairing of parts of this chromosome and association with the sex chromosome complex as a possible mediator of the biased segregation. We also discovered polymorphic polyalanine tract expansions in platypus SOX3 with some bias of longer expansions in females and diversified expansions in males possibly as a result of the non-random segregation at meiosis. These results raise the possibility that the therian sex chromosomes commenced differentiation before the monotreme-therian mammal divergence and was replaced in monotremes by a different sex chromosomes system. This work also indicates that non-random segregation could be an early step in autosome to sex chromosome differentiation.