The identification of the sex chromosomes in the three extant species of Prototherian mammals (the monotremes) is complicated by their involvement in a multivalent translocation chain at the first division of male meiosis. The platypus X chromosome, identified by the presence of two copies in females and one in males, has been found to possess a suite of genes that have been mapped to the X chromosomes of all eutherian and metatherian mammals. We have extended gene mapping studies to a member of the only other extant monotreme family, the echidna, which has a G-band equivalent X1 chromosome, as well as a smaller X2. We find that the five human X-linked genes (G6PD, GDX, F9, AR and MCF2) map to the echidna X1 chromosome in locations equivalent to those on the platypus X. These results confirm that the echidna X1 is the original X chromosome in this species, and identify a conserved ancestral monotreme X chromosome.