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Brucellosis is a bacterial zoonosis worldwide and a potential health risk in Pakistan due to socio-economic reasons and lack of awareness, veterinary health and modern dairy-processing facilities. The aim of this study was to determine the burden of brucellosis in hospitalized patients of selected districts of Punjab, Pakistan. To this end, a total of 280 serum samples were collected from suspected patients in districts Lahore, Okara and Khushab between 2016 and 2018. Anti-Brucella antibodies were detected in 89 (31.8%) sera by Rose Bengal plate test (RBPT), and Brucella abortus DNA was amplified by polymerase chain reaction (PCR) in 42 (15%) samples. In univariable analysis, the seroprevalence varied significantly (p < 0.05) between districts with Lahore indicating 41/101 (40.6%) Odds ratio (OR) = 53.98 (7.22–403.67; 95% CI), Okara 47/99 (47.5%) OR = 71.4 (9.55–533.65; 95% CI) and Khushab 1/80 (1.3%) as reference. Similarly, exposure to aborted materials and a history of blood transfusion did associate significantly (p < 0.05), posing OR = 2.91 (1.58–5.36; 95% CI) and OR = 4.48 (2.56–7.84; 95% CI) risk. However, consumption of raw milk and history of blood transfusion did associate significantly (p < 0.05) in multivariable analysis indicating OR = 1.91 (1.09–3.34; 95% CI) and OR = 4.92 (2.76–8.75; 95% CI), respectively. The association of history of blood transfusion as a brucellosis risk factor underscores the need to create brucellosis awareness programs both at public and institutional levels in the medical and veterinary fields, as well as facilities and capacity building to delve deeper into the epidemiology of brucellosis in the country. Particular attention should be paid to chronically infected patients. Therefore, laboratory modernization, standardization and validation of the diagnostic tests, and staff trainings are required. Finally, pasteurization of the milk is recommended before consumption. [ABSTRACT FROM AUTHOR]