We report the DNA sequences of protamine P1 genes and flanking regions from 21 mammalian species, including representatives of all extant marsupial orders. The protamine P1 locus in marsupials displays highly conserved 5' and 3' flanking sequences, as well as a highly variable intron. Marsupial protamines examined are distinct from those of eutherian mammals in lacking cysteine residues, a feature that may be correlated with lower stability of marsupial sperm nuclei during chromatin condensation. Phylogenetic analysis of protamine sequences leads to the following conclusions: (i) the microbiothere Dromiciops is part of a clade that includes dasyurids, Notoryctes and diprotodontians but not bandicoots; (ii) dasyurids are the closest living relatives of Notoryctes; (iii) macropodids, phalangerids and pseudocheirids form a clade apart from the phascolarctids; and (iv) the closest living relatives of caenolestids are didelphids.