The homeobox genes comprise a large and diverse gene superfamily, many of which encode transcription factors with pivotal roles in the embryonic development of animals. We searched the assembled draft genome sequence of an amphioxus, Branchiostoma floridae, for genes possessing homeobox sequences. Phylogenetic analysis was used to divide these into gene families and classes. The 133 amphioxus homeobox genes comprise 60 ANTP class genes, 29 PRD genes (excluding Pon and Pax1/9), nine TALE genes, seven POU genes, seven LIM genes, five ZF genes, four CUT genes, four HNF genes, three SINE genes, one CERS gene, one PROS gene, and three unclassified genes. Ten of the 11 homeobox gene classes are less diverse in amphioxus than humans, as a result of gene duplication on the vertebrate lineage. Amphioxus possesses at least one member for all of the 96 homeobox gene families inferred to be present in the common ancestor of chordates, including representatives of the Msxlx, Bari, Abox, Nk7, Ro, and Repo gene families that have been lost from tunicates and vertebrates. We find duplication of several homeobox genes in the cephalochordate lineage (Mnx, Evx, Emx, Vent, Nk1, Nedx, Uncx, Lhx2/9, Hmbox, Pou3, and Irx) and several divergent genes that probably originated by extensive sequence divergence (Hx, Ankx, Lcx, Acut, Atale, Azfh, Ahbx, Muxa, Muxb, Aprd1-6, and Ahnf). The analysis reveals not only the repertoire of amphioxus homeobox genes but also gives insight into the evolution of chordate homeobox genes.