In the present study, three Y-box cDNAs, named as Lyb1, Lyb2, and Lyb3, were cloned from the liver and oral gland of Japanese lamprey (Lampetra japonica) using reverse transcript-PCR (RT-PCR) and rapid amplification of cDNA ends (RACE) techniques. Further bioinformatical analysis indicated that the three Y-box genes of lamprey, i.e., Lyb1, Lyb2, and Lyb3, encoded 331, 181, and 171 amino acids, respectively. The three amino acid sequences were homologically aligned with those from Ciona intestinalis, Danio rerio, Oryzias latipes, Takifugu rubripes, and Mus musculus by DNAMAN software. The Lyb genes were novel members of Y-box gene family containing a conserved cold-shock domain with two RNA binding motifs (RNP-1 and RNP-2). Moreover, phylogenetic analysis revealed that there was only one type of Y-box gene before the emergence of gnathostomata. By contrast, the Y-box gene were divided into three groups, YB1, YB2, and CSDA during evolution of jawed vertebrates.