Introduction: Trypanosoma caninum is a protozoan species recently described in dogs, whose occurrence has been reported in areas of overlap with visceral leishmaniasis.
Methods: Trypanosoma sp. were isolated from nine dogs and characterized by molecular methods.
Results: PCR and sequencing confirmed the presence of T. caninum in all dogs, revealing two new areas of transmission: Barra Mansa and São João do Piauí.
Conclusions: The nine new cases described, when added to those already published, account for 62 cases of natural infection by T. caninum and show the geographical spread of this species to new areas, where other trypanosomatids circulate.