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Prevention and control mechanisms against different virus infections in poultry species is one of the biggest concern present in poultry industry. The continuous evolving nature and frequent mutation characteristics of viruses, especially avian influenza virus, is the biggest concern. This is due to their ability to cause disease and mortality in both poultry and humans. Limitations of the available preventive measures against rapidly evolving strains of the viruses suggest the requirement of new prophylactic measures. Pattern recognition receptors, mainly Toll-like receptors (TLRs), in the host body are responsible for recognizing virus infections. Activation of TLRs has been found to reduce the replication of the viruses within the host. Toll-like receptor 3 (TLR3) is a crucial member of the TLR family. Specificity of the TLR3 to recognize dsRNA motifs of pathogens, a universal marker in the virus infections, indicate its importance in virus infections and immunity induction in the host. Study of TLR3, along with its alternative splicing and mechanism involved in its activation, has mainly been reported in human and mouse. However, little information is available about TLR3 in avian species and no study has been done specifically directed to Japanese quail (Coturnix japonica). Thus, it is important to characterize the TLR3 gene of avian species for investigating their role in prevention of the viral replication. We determined the coding sequence of quail TLR3 for the first time and examined the alternative splicing isoforms of TLR3 gene in chicken, turkey and quail. Three TLR3 alternative isoforms were identified in quail and turkey whereas only full length TLR3 present in chicken. TLR3-A produced full-length proteins in all the avian species. In quail, there was deletion of 275 and 205 nucleotides in qTLR3-B and qTLR3-C respectively, whereas turkey tTLR3-B and tTLR3-C isoforms had deletion of 275 and 396 nucleotides. Deletion of the nucleotides caused development of a premature stop codon and thus produced a small protein. Expression of TLR3, along with its alternative spliced isoforms was detected in most of the tested tissues suggesting ubiquitous expression in the avian species cells and tissues. In addition, the cleavage peptide at C terminal ectodomain, reported to undergo proteolytic processing in mammal TLR3 activation, was found to be absent in the avian species. Absence of the cleavage peptide sequence and the absence of cleaved fragment of quail TLR3 protein in Western blot suggests the absence of cleavage activity in the C terminal ectodomain of the avian TLR3. Alternative spliced isoforms of TLR3 in avian species may have a specific role, and importance, in signaling mechanism. Similarly, absence of cleavage activity suggests a difference in the activation mechanism, in avian TLR3 compared to its mammalian counterpart. The information obtained from this characterization study of the avian TLR3 gene could be helpful in exploring the role of avian TLR3 along with its isoforms, in virus infections.