In this cross-sectional study, a multistage sampling technique was used to estimate the seroprevalence and risk factors of brucellosis in small ruminant (sheep and goats) populations of Punjab, Pakistan. It was also aimed to identify and differentiate Brucella species from serum samples of sheep and goats. A total of 1783 serum samples from 219 holding premises of small ruminants across three districts (Chakwal, Dera Ghazi Khan, and Jhang) of Punjab province were collected and subjected to RBT and commercially available indirect ELISA kits with a simultaneous screening approach. The flock-based seroprevalence was estimated as 9.13% (95% CI: 5.9-13.7) with a statistically higher prevalence of 17.5% (95% CI: 10.0-28.6) in flocks with ≥50 animals, and linear regression model showed the significant linear association (F= 5.74; p= 0.01) between number of animals in a flock and seroprevalence. At the animal level, the apparent seroprevalence was estimated as 0.6% (CI 95%: 0.3-1.1) by RBT and 2.0% (CI 95%: 1.5-2.8) by the ELISA test. Based on the ELISA test, the true prevalence of brucellosis at the animal level was calculated as 1.6% (CI 95%: 1.0-2.3). The results from the logistic regression model showed that animal pregnancy status and abortion history are associated with brucellosis and have significantly higher odds ratios of 3.04 (95% CI, 1.31-7.03) and 2.71 (95% CI, 1.23-5.95) respectively. The geographical distribution of brucellosis was limited to some villages with intra-cluster/flock coefficient ICC (ρ) of 0.26 in small ruminant flocks. The quantitative real-time PCR (qRT-PCR) identified brucella genus-specific and B. abortus DNA from serum samples with none of the samples positive for B. melitensis. The parallel interpretation of screening tests found agreement between RBT and ELISA tests at a fair level (kappa: 0.37, 95% CI: 0.20-0.55). The study contributed to the existing epidemiological information on brucellosis in small ruminants
