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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), p = 0.00], location in urban area [OR: 2.99 (95% CI: 1.46–6.51), p = 0.00],body condition [OR: 3.47 (95% CI: 1.64–7.99), p = 0.00], presence of farcy lesion[OR: 7.71 (95% CI: 3.47–19.50), p = 0.00], proximity to water bodies [OR: 7.71 (95% CI: 3.47–19.50), p = 0.00]; domestic animal population [OR: 3.20 (95% CI: 1.24–10.87), p = 0.03] and number of households in sampling area [OR: 4.18 (95%CI: 1.82–11.30), p = 0.00]. The study provides an estimate of prevalence of glanders and a potential link between animal seropositivity and presence of B. mallei in soil. The risk factors identified in this study can be used in surveillance and disease awareness. The high prevalence of disease in draught horses and contact of infected animals with their care-takers in developing countries signify need to initiate progressive control of the disease using one health approach. [ABSTRACT FROM AUTHOR]