The Zinc Fingers and Homeoboxes (Zhx) proteins, Zhx1, Zhx2, and Zhx3, comprise a small family of proteins containing two amino-terminal C2-H2 zinc fingers and four or five carboxy-terminal homeodomains. These multiple homeodomains make Zhx proteins unusual because the majority of homeodomain-containing proteins contain a single homeodomain. Studies in cultured cells and mice suggest that Zhx proteins can function as positive or negative transcriptional regulators. Zhx2 regulates numerous hepatic genes, and all three Zhx proteins have been implicated in different cancers. Because Zhx proteins contain multiple predicted homeodomains, are associated with interesting physiological traits, and seem to be only present in the vertebrate lineage, we investigated the evolutionary history of this small family by comparing Zhx homologs from a wide range of chordates. This analysis indicates that the zinc finger motifs and homeodomains are highly similar among all Zhx proteins and also identifies additional Zhx-specific conserved regions, including a 13 amino acid amino-terminal motif that is nearly identical among all gnathostome Zhx proteins. We found single Zhx proteins in the sea lamprey (Petromyzon marinus) and in the nonvertebrate chordates sea squirt (Ciona intestinalis) and lancelet (Branchiostoma floridae); these Zhx proteins are most similar to gnathostome Zhx3. Based on our analyses, we propose that a duplication of the primordial Zhx gene gave rise to Zhx3 and the precursor to Zhx1 and Zhx2. A subsequent tandem duplication of this precursor generated Zhx1 and Zhx2 found in gnathostomes.