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Background and Aim: Canine monocytic ehrlichiosis is a vector-borne disease caused by the obligatory intracellular bacterium Ehrlichia canis, which is distributed across tropical and subtropical regions worldwide. Its prevalence within dog populations is high in municipalities located across the Pantanal biome, but it remains unknown in Barão de Melgaço, Mato Grosso, Brazil. This study aimed to determine the molecular prevalence and factors associated with E. canis infection in dogs domiciled in Barão de Melgaço. Materials and Methods: A cross-sectional study was carried out to investigate the prevalence of E. canis infection in 369 dogs from urban and rural areas in Barão de Melgaço, North Pantanal wetland, Brazil. Initially, the dogs were examined, and, through a questionnaire, the risk factors were investigated. Blood samples were subjected to DNA extraction and PCR was performed to estimate the prevalence of E. canis infection. Results: The molecular prevalence of E. canis infection in dogs was 42.5% and none of the studied variables were significantly associated with polymerase chain reaction (PCR) positivity (p > 0.05). Conclusion: The high molecular prevalence demonstrates an increased transmission of the agent across the city. This also indicates that attention needs to be paid to E. canis infection and control measures should be introduced to prevent its transmission. The demographic and clinical risk factors commonly associated with E. canis infection in this study were not associated with PCR positivity. [ABSTRACT FROM AUTHOR]