Abstract: This cross sectional study was designed to determine the current status and prevalence of brucellosis in cattle and buffaloes. A total of 1220 animals (895 cows, 325 buffaloes) from the five agro-climatic regions of Punjab were screened for brucellosis using rose bengal plate agglutination (RBPT) and microtiter plate agglutination test (MAT), from April 2012 to May 2013. In the study population, an overall seroprevalence of brucellosis was observed as 27.95 and 18.11% by RBPT and MAT, respectively. Statistically significant (p<0.01) differences in prevalence of brucellosis among cattle and buffaloes were 23.01% (206/895), 18.21% (163/895) and 30.99% (84/325), 19.92% (54/325) by RBPT and MAT, respectively. Among the two tests applied in this present study, RBPT was found to be more sensitive than MAT.