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Leptospirosis is a worldwide emerging infectious disease of zoonotic importance and large epidemics and epizootics have been reported all over the globe. A cross survey study was conducted to estimate seroprevalence of human leptospirosis in climatically distinct regions of Pakistan and to identify the risk factors associated with the disease. Blood samples from 360 humans were collected through convenient sampling, 120 from each of three study areas. Serological testing was performed using ELISA kit as per manufacturer’s recommendations. The results showed an overall prevalence of 40.83% (95% CI; 35.71-46.11). Statistical analysis showed significant (P < .05) differences in leptospiral seroprevalence in three different geographic locations, with highest in humid sub-tropical climatic region (50.83%; 95% CI; 41.55-60.07), followed by semi-arid region (44.16%; 95% CI; 35.11-53.52) and lowest in hot and dry region (27.50%; 95% CI; 19.75-36.40). After multivariate analysis age, gender, exposure to flooding water, source of water usage, disinfection schedule of surroundings and history of cut and wound were found significantly associated with the seropositivity of Leptospira. The present study, first to uncover seroprevalence of human Leptospira in different climatic regions of Pakistan, alarms about effect of climate on prevalence of Leptospira in the region.