The homeobox is a relatively conserved 180-nucleotide-long sequence characterizing a large and phylogenetically wide-spread family of putative regulatory genes, many of which have been implicated in the control of embryonic development. The Drosophila melanogaster muscle segment homeobox (msh) gene contains a homeobox which is markedly divergent from that of any other characterized D. melanogaster gene, yet very similar to that of the mouse Hox-7.1 gene [Robert et al., EMBO J. 8 (1989) 91-100; Hill et al., Genes Develop. 3 (1989) 26-37]. To elucidate the evolutionary relationship between these two genes, and hence facilitate comparative analyses, it is necessary to determine the number of msh-like genes in the two species, and to characterize any msh gene duplication events which may have occurred since their divergence. To address these questions, I have employed the polymerase chain reaction to amplify, clone and sequence msh-related homeobox genes from two divergent vertebrate species (mouse and zebrafish), one ascidian (Ciona intestinalis), and D. melanogaster. Sequence determination of 91 recombinant clones resulted in the identification of three msh-like genes from mouse and three from zebrafish, but only one each from C. intestinalis and D. melanogaster. Sequence analyses suggest that multiple msh gene duplications occurred in the lineage leading to vertebrates, after the divergence of the ascidia.