Cathelicidin synthesized by bone marrow cells plays an important role in neutralizing invading pathogens. In the present study, the myeloid cathelicidin cDNA from Bubalus bubalis has been cloned and characterized. RNA from bone marrow of buffalo ribs was extracted, reverse transcribed and amplified using specific pair of primer designed from published cathelicidin-4 cDNA sequences of Bos taurus popularly known as indolicidin. An expected amplified product of 517 bp was obtained, which was cloned and sequenced. Comparison of buffalo cathelicidin and indolicidin sequences reveal that the open reading frames (ORF) of both these two congeners consist of 435 nucleotides with 28 divergent nucleotides and the translated proteins of 144 amino acid residues. Fourteen amino acid residues were found to be dissimilar between these two congeners. The molecular mass of buffalo cathelicidin calculated from the deduced amino acid sequence was 16.23 kDa, which is in close proximity of indolicidin. The sequence comparison with known B. taurus cathelicidin congeners again show 70.8-92.9% identity at nucleotides level and 65-88.3% identity at amino acids level. The maximum similarity of buffalo cathelicidin both at nucleotides level (92.9%) and protein level (88.3%) was found to be with indolicidin. Phylogenetic tree analysis at nucleotides and amino acids level indicate that buffalo, cattle, sheep, pig and equine cathelicidin sequences comprise one clade which are distantly related with human, rabbit and murine cathelicidins. It may be reasonably concluded that buffalo possess the ancestral gene of cathelicidin like that of bovine species.