Gnathostomes have two lineages of Otx genes, Otx1 and Otx2, as cognates of a Drosophila head gap gene, orthodenticle. Previous studies with mutant mice have demonstrated that they play essential roles in the development of rostral head. To shed lights on the evolution of the rostral head in vertebrates we isolated their cognates in the Japanese marine lamprey, Lampetra japonica. The lamprey genome appeared to have two Otx cognantes, LjOtxA and LjOtxB. Phylogenetic analyses suggest that LjOtxA clusters with gnathostome Otx2 genes, but LjOtxB does not belong to either the Otx1 or Otx2 lineage. LjOtxA was expressed in the forebrain and midbrain with the caudal limit possibly at the midbrain/hindbrain junction as gnathostome Otx cognates are, but LjOtxB was not expressed in the brain. No Otx1 or Otx2 cognates are known in gnathostomes that are not expressed in the brain. Both LjOtxA and LjOtxB were expressed in the olfactory placode, epiphysis, optic stalks, and lower and upper lips. LjOtxB was also expressed in the eyes, where no LjOtxA transcripts were detected. Thus, Otx1 and Otx2 functions for the development of forebrain and midbrain in gnathostomes appear to be shouldered by LjOtxA alone in the lamprey. LjOtxB may have diverged from the stem of the Otx1 and Otx2 lineages and evolved independently.