The complete coding sequence (CDS) of water buffalo Annexin A9 (ANXA9) was amplified and identified using the reverse transcription-polymerase chain reaction (RT-PCR) based on the conserved sequence information of cattle or the expressed sequence tags (ESTs) of other Bovidae species. Sequence analysis revealed that the CDS of water buffalo ANXA9 encodes an enzyme of 345 amino acid residues with a deduced molecular weight of 38.45 kDa and a PI of 8.74. The deduced amino acid sequence of water buffalo ANXA9 shares 98, 83, 86.4, 76.5, 77, 82, 83.8 and 45.7% identity with its homologous sequences of cattle, horse, pig, mouse, rat, chimpanzee, human, African clawed frog respectively. The phylogenetic tree analysis based on the CDS of ANXA9 gene showed that water buffalo has a closer genetic relationship with cattle than with other species. The ANXA9 was widely expressed in the buffalo tissues examined, being high in the pituitary gland, muscle and brain; moderate in the heart, spleen, liver, mammary gland and lung; weakly expressed in the small intestines and skin and not expressed in the adipose tissue and stomach. The results of the present study will establish a foundation for further insights into this novel water buffalo gene.