Abstract Two or more isoforms of several cytokines including tumor necrosis factors (tnfs) have been reported from teleost fish. Although zebrafish (Danio rerio) and medaka (Oryzias latipes) possess two tnf-α genes, their genomic location and existence are yet to be described and confirmed. Therefore, we conducted in silico identification, synteny analysis of tnf-α and tnf-n from both the fish with that of human TNF/lymphotoxin loci and their expression analysis in zebrafish. We identified two homologs of tnf-α (named as tnf-α1 and tnf-α2) and a tnf-n gene from zebrafish and medaka. Genomic location of these genes was found to be as: tnf-α1, and tnf-n and tnf-α2 genes on zebrafish chromosome 19 and 15 and medaka chromosome 11 and 16, respectively. Several features such as existence of TNF family signature, conservation of genes in TNF loci with human chromosome, phylogenetic clustering and amino acid similarity with other teleost TNFs confirmed their identity as tnf-α and tnf-n. There were a constitutive expression of all three genes in different tissues, and an increased expression of tnf-α1 and -α2 and a varied expression of tnf-n ligand in zebrafish head kidney cells induced with 20μgmL−1 LPS in vitro. Our results suggest the presence of two tnf-α homologs on different chromosomes of zebrafish and medaka and correlate this incidence arising from the fish whole genome duplication event.