In Brazil, although the domestic dog is a major target for the control actions for visceral leishmaniasis, knowledge gaps of the Leishmania species present in those animals still exist in many endemic areas. The objective of this study was the use of parasitological culture as a diagnosis tool and identification of species of Leishmania and other trypanosomatids in the canine population in the city of Cuiaba/Mato Grosso. Biological samples such as blood, intact skin fragments, cutaneous ulcers, and bone marrow were collected during a cross-sectional study and cultured on biphasic medium (Novy-MacNeil-Nicolle [NNN]/Schneider's). Leishmania isolates were characterized through isoenzyme electrophoresis. Isolates were obtained from 11.2% (n=54) of the 482 animals studied considering the different anatomical sites investigated. Leishmania chagasi was confirmed in 8.3% (n=40) dogs and Trypanosoma caninum in 2.9% (n=14). The sample of intact skin presented a higher chance of isolation of L. chagasi in symptomatic dogs and bone marrow in asymptomatic dogs (p<0.05). The results presented in this study emphasize the value of culture and confirm, for the first time, the circulation of L. chagasi in the canine population in different neighborhoods of the city of Cuiaba and broaden the knowledge of the geographical distribution of T. caninum in Brazil.