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Glanders is an infectious and contagious bacterial disease of equines. A little is known about its seroprevalence and risk factors in working equines in countries where the disease is endemic. Also, there are no reports on prevalence of the disease in areas where there is a prior evidence of Burkholderia (B.) mallei detection in soil. A cross-sectional study was conducted in selected districts (n = 09) of Punjab province of Pakistan during 2014–2015. A total of 1008 serum samples were screened for detection of antibodies to B. mallei with complement fixation test followed by western blot. The overall seroprevalence was found to be 3.17% (95% CI: 2.25–4.44). The seropositivity was significantly higher from the sampling sites where B. mallei was detected in soil [OR: 10.66 (95% CI: 4.42–31.66), p = 0.00]. Other risk factors significantly associated with animal seropositivity were: age group [OR: 1.78 (95% CI: 4.58–15.56 …