Haemotropic mycoplasmas are pleomorphic, epicellular, gram-negative bacteria, which infect red blood cells. In dogs, the acute phase of this infection may lead to haemolytic anaemia potentially precipitated by splenectomy, or by immunosuppressive drugs or diseases; however, in cases of chronic infection, the signs are inapparent. Molecular techniques and microscopic observation are the most commonly used methods to diagnose mycoplasma infection, though studies about haemotropic mycoplasma infections in dogs from Cuiabá, capital of Mato Grosso, Brazil are scarce. The objective of this study was to evaluate the frequency of mycoplasma infection, the Mycoplasma species present, and factors associated with infection in 334 dogs diagnosed using polymerase chain reaction (PCR). The frequency of infection was 5.4%, and M. haemocanis was the only species identified. No epidemiological factor investigated showed a statistically significant association with infection, including tick infestation.