We sequenced the protamine P1 gene (ca. 450 bp) from 20 bats (order Chiroptera) and the flying lemur (order Dermoptera). We compared these sequences with published sequences from 19 other mammals representing seven orders (Artiodactyla, Carnivora, Cetacea, Perissodactyla, Primates, Proboscidea, and Rodentia) to assess structure, base compositional bias, and phylogenetic utility. Approximately 80% of second codon positions were guanine, resulting in protamine proteins containing a high frequency of arginine residues. Our data indicate that codon usage for arginine differs among higher mammalian taxa. Parsimony analysis of 40 species representing nine orders produced a well-resolved tree in which most nodes were supported strongly, except at the lowest taxonomic levels (e.g., within Artiodactyla and Vespertilionidae). These data support monophyly of several taxa proposed by morphologic and molecular studies (all nine orders: Laurasiatheria, Cetartiodactytla, Yangochiroptera, Noctilionoidea, Rhinolophoidea, Vespertilionoidea, Phyllostomidae, Natalidae, and Vespertilionidae) and, in agreement with recent molecular studies, reject monophyly of Archonta, Volitantia, and Microchiroptera. Bats were sister to a clade containing Perissodactyla, Carnivora, and Cetartiodactyla, and, although not unequivocally, rhinolophoid bats (traditional microchiropterans) were sister to megachiropterans. Sequences of the protamine P1 gene are useful for resolving relationships at and above the familial level in bats, and generally within and among mammalian orders, but with some drawbacks. The coding and intervening sequences are small, producing few phylogenetically informative characters, and aligning the intron is difficult, even among closely related families. Given these caveats, the protamine P1 gene may be important to future systematic studies because its functional and evolutionary constraints differ from other genes currently used in systematic studies.