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This study investigated the risk factors associated with seroreactivity to Leptospira sp. in horses from Brazilian Amazon. Blood samples were collected and microscopic agglutination test was performed, and univariate and multivariate analyses were conducted for association with risk factors. General seroprevalence was 32.7% and the most prevalent serogroup observed was Sejroe (5.9%). Seroreactivity was associated with age, sex, activity (work, leisure, or both), feed supplementation, trash accumulation, and presence of felines. Epidemiology of leptospirosis remains poorly understood in Amazon region, particularly in livestock, requiring more studies to improve control and prevention measures.High seroreactivity for Sejroe was observed in horses from Brazilian Amazon.Diversity of serogroups found potentially related to host diversity in environment.Epidemiology of leptospirosis remains understood in Amazon region.