Canine babesiosis is a common haemoparasitosis in Brazil. Caused by parasites of the genus Babesia, it is transmitted by ixodid ticks and affects domestic and wild canids. The objective of this study was to verify the prevalence of Babesia species (spp.) using molecular methods in dogs living in urban and rural areas of Cuiabá, Mato Grosso State, Brazil, and to identify the main factors associated with infection. A total of 407 samples from 407 dogs were evaluated using a polymerase chain reaction (PCR) technique, among which Babesia species (spp.) was amplified in 10 (2.5%). Although, no statistical association was found among the variables studied (p>0.05), greater positivity was observed in dogs<1 year of age, male sex, those with free access to the street, and the presence of ticks. PCR samples positive for Babesia spp. were submitted to sequencing and compared in GenBank and exhibited a high degree of similarity with Babesia vogeli sequences.