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Glanders is highly contagious and zoonotic bacterial disease of equines caused by Burkholderia mallei. Since many decades, glanders is endemic in eastern parts (Punjab) of Pakistan and has no information of disease in the north-western region (Khyber Pakhtunkhwa – KPK). Therefore, present study was aimed to determine the seroprevalence and associated risk factors in two draft equine populated districts (Charsada and Peshawar) of KPK. A total of 393 equine samples originating from different localities of study area were investigated for anti-B. mallei antibodies (glanders) using indirect enzyme linked immunosorbent assay (ID Vet, Grabels France). The overall seroprevalence was found to be 3.1% (95% CI: 1.6-5.3) which remained insignificantly (χ2 0.421, P=0.516) between the two districts. In Charsada, prevalence estimates varied significantly (χ2 11.986, P=0.017) among 5 localities and found insignificant (χ2 1.287, P=0.732) in different sampling areas of Peshawar district. Donkeys were more seropositive (4.1%; 4/195) than horses (2.1%; 8/194) which did not differ statistically (χ2 1.397, P=0.237) and all mules were tested negative. Prevalence slight increased with age of animals (>10 years) and with poor body conditions. Donkeys were more test positive (OR: 2.05, 95% CI: 0.61-6.94) than horses and similarly animals managed on communal feeding (OR: 1.84, 95% CI: 0.23-14.5) and water troughs (OR: 1.65, 95% CI: 0.21-13.04) had more chances of contracting glanders. Multivariable model indicated donkeys (OR: 4.58, 95% CI: 1.0, 20.9) and equines above 10 years of age (OR: 3.10, 95% CI: 0.7-13.3) were more likely to test positive for glanders. In conclusion, present study provides prevalence estimates of equine glanders in KPK regions, which appears to be an area of endemicity. Future large-scale investigations are seriously warranted in other equine populated districts before implementing an effective control plan for glanders. [ABSTRACT FROM AUTHOR]