Environment influences the composition, distribution, and behavior of the vectors and mammalian hosts involved in the transmission of visceral leishmaniasis (VL), affecting the epidemiology of the disease. In Brazil, the urbanization process and canine cases of VL are indicators for local health authorities. This study aimed to investigate the occurrence of the canine visceral leishmaniasis (CVL) in Maranhão State, Brazil. Blood samples collected from 960 dogs from six municipalities and six different ecosystems (Baixada Maranhense, Mangue, Mata dos Cocais, Amazônia, Cerrado, and Restinga) to serological tests (enzyme-linked immunosorbent assay [ELISA], indirect fluorescence antibody test [IFAT], and chromatographic immunoassay methods [Dual Path Platform technology, DPP(®)]) and parasitological diagnosis. From serological tests, 11.14% (107) of the dogs were positive for CVL, with 59.16% (568), 14.5% (148), and 131% (126) positives to ELISA, DPP, and IFAT tests, respectively. Only seven animals (0.73%) were positive in a parasitological test. We also performed parasite isolation and phylogenetic characterization. All isolates of dogs obtained from Maranhão were grouped in a single branch with Leishmania infantum chagasi from Brazil. The ecosystem Amazonia presented the highest positivity rates to CVL in serological and parasitological tests. Brazilian biomes/ecosystems suffer large degradation and may favor, depending on climatic conditions, the installation of new diseases. In the case of VL, dogs are reservoirs of parasites and sentinels for human infection.