Bilaterian Hox genes play pivotal roles in the specification of positional identities along the anteroposterior axis. Particularly in vertebrates, their regulation is tightly coordinated by tandem arrays of genes [paralogy groups (PGs)] in four gene clusters (HoxA-D). Traditionally, the uninterrupted Hox cluster (Hox1-14) of the invertebrate chordate amphioxus was regarded as an archetype of the vertebrate Hox clusters. In contrast to Hox1-13 that are globally regulated by the “Hox code” and are often phylogenetically conserved, vertebrate Hox14 members were only recently revealed to be present in an African lungfish, a coelacanth, chondrichthyans and a lamprey, and decoupled from the Hox code. In this study we performed a PCR-based search of Hox14 members from diverse vertebrates, and identified one in the Australian lungfish, Neoceratodus forsteri. Based on a molecular phylogenetic analysis, this gene was designated NfHoxA14. Our real-time RT-PCR suggested its hindgut-associated expression, previously observed also in cloudy catshark HoxD14 and lamprey Hox14α. It is likely that this altered expression scheme was established before the Hox cluster quadruplication, probably at the base of extant vertebrates. To investigate the origin of vertebrate Hox14, by including this sarcopterygian Hox14 member, we performed focused phylogenetic analyses on its relationship with other vertebrate posterior Hox PGs (Hox9-13) as well as amphioxus posterior Hox genes. Our results confirmed the hypotheses previously proposed by other studies that vertebrate Hox14 does not have any amphioxus ortholog, and that none of 1-to-1 pairs of vertebrate and amphioxus posterior Hox genes, based on their relative location in the clusters, is orthologous. J. Exp. Zool. (Mol. Dev. Evol.) 316:515–525, 2011. © 2011 Wiley Periodicals, Inc.