Brucellosis is a worldwide zoonosis and a significant cause of loss of health in human and animals. Conventionally, definitive diagnosis is done by isolation of brucellae which is time consuming, technically demanding and may be hazardous. The present study was aimed to develop PCR assay based on evolutionarily conserved outer membrane protein 22 gene for rapid diagnosis of Brucella spp. A set of primer was designed after multiple alignment of outer membrane protein 22 gene sequences by clustalW. PCR amplification resulted in single specific amplicon of 639 bp in all the Brucella strains tested. The primers were found to be highly sensitive and specific. PCR assay developed may be used for clinical diagnosis of Brucella infection.