A number of immune-related signal molecules, cytokines and chemokines, have been identified in fish complete genome sequence by in silico analyses. However, the information on these molecules is still limited in aquaculture species such as Japanese flounder, Paralichthys olivaceus. Here, we performed expressed sequence tag analysis using illumina sequencing technology and identified three unique sequences homologous to fish Interleukin-1β (IL-1β), two of which were distinct from the Japanese flounder IL-1β (jfIL-1β). IL-1β is a well-conserved inflammatory cytokine, and has multiple functions in the host defense. The novel IL-1β homologues were designated as novel IL-1β-1 and -2 (nIL-1β-1 and -2). Although jfIL-1β mRNA encodes 246 amino acid residues, deduced amino acid sequences of nIL-1β-1 and -2 were composed of 262 and 354 residues, respectively. nIL-1β-1 and -2 were most homologous to Arctic char IL-1β and to the predicted interleukin-1 receptor antagonist of rainbow trout, respectively. The genomic sequence of nIL-1β-1 and -2 are spanned by 5 and 6 introns, respectively, while jfIL-1β is divided by 3 introns. It is thus speculated that the genes might have not evolved from a common ancestral gene but individually. Both of the nIL-1β transcripts were detected in many tissues. Interestingly, these genes were not up-regulated by pathogenic cell treatment, which induces jfIL-1β expression in kidney and spleen. Meanwhile, nIL-1β-2 was slightly induced in peripheral blood leukocytes by in vitro lipopolysaccharide treatment.