Brucellosis is an emerging animal as well as human health issue in Pakistan. Population based estimation of true prevalence is time occurring in different livestock species and requires to develop prevention and control strategy, that hasn't been done in Pakistan. Active disease surveillance at door steps was conducted to collect blood samples from small and large ruminants of subtropical rural communities in the province of Punjab during January-March, 2015. Total 59665 sera samples from apparently healthy animals were collected and screened by Rose Bengal Plate test and confirmed by Complement fixation test, respectively. After adjustment for test sensitivities and specificities accordingly, and chi square analysis of results, the true prevalence per 1000 animals in buffaloes was (7.86%), significantly (P<0.01) higher than small ruminants (5.84%) and cattle (2.05%). Being a subtropical region, geographically significant (P<0.01) variation was observed in burden and pattern of brucellosis i.e., the highest prevalence was in Central (14.81%) followed by South (0.5%) and northern (0.4%) Punjab. Considering the high mean prevalence and its zoonotic importance it can be devastating if not controlled because of bad management practices, lack of awareness and close contact of farmers with their livestock in Pakistan. Where the socio-economic conditions of rural population does not allow test and slaughter policy, therefore, mass vaccination is recommended to control brucellosis through local government support.