The central region of Brazil is known to be an endemic area for canine ehrlichiosis. Therefore, this study aims to determine the prevalence rates of E. canis infection in dogs and in Rhipicephalus sanguineus ticks collected from the dogs and their home environments. Serum samples and genomic DNA from the blood of 20 dogs and 299 ticks were analyzed by IFA and PCR assays in order to detect Ehrlichia canis antibodies and DNA. Nine (45%) of the 20 dogs were seropositive for E. canis, with titers ranging from 80 to 10240, and 6 dogs (30%) were positive for Ehrlichia spp. by PCR. Five free-living ticks were positive (2.89%, 95% confidence interval: 0.94-6.62%), as were six ticks attached to dogs (4.76%; 95% CI: 1.77-10.0%). The two groups showed a similar infection rate (P=0.395). Partial dsb DNA sequences of two samples from ticks were identical to each other and 100% (350/350 nucleotides) were identical to E. canis. Despite the high serological and molecular rates of canine ehrlichiosis in Cuiabá, the prevalence among infected ticks was lower than that found among dogs. However, adult ticks may remain infective much longer to ensure their infestation and infection of susceptible dogs.