The three extant genera of the prototherian mammals, Ornithorhynchus (platypus), Tachyglossus (Australian echidna) and Zaglossus (New Guinea echidna), all have a mechanism of sex determination at odds with that seen in eutherian and metatherian mammals. Indeed, they stand apart from all vertebrates. Instead of the XX/XY, X₁X₂Y or ZZ/ZW systems seen in the majority of vertebrates the monotremes have a chain of nine (or ten) chromosomes present during meiosis in the male. This is believed to be the consequence of a presumed series of reciprocal translocations involving four autosomal pairs and the original X and Y chromosomes. The presence of this chain in all three genera indicates that a similar chain occurred in their common ancestor. This paper provides an overview of the search to unravel the mystery of this chain and to determine the identity of the sex chromosomes and members of the chain. The development of new techniques has hugely facilitated clarification of the findings of the earlier researchers. As a result, the chromosomes of the platypus and the echidna have now been individually described, the chain elements and/or sex chromosomes have been identified unambiguously and their order in the chain has been determined. The research reviewed here has also provided insights into the evolution of mammalian sex chromosomes and given new directions for unravelling dosage compensation and sex-determination mechanisms in mammals.