In Brazil, the parasite associated with human intestinal schistosomiasis is Schistosoma mansoni, which has also been found infecting wild rodent species. The municipality of Sumidouro is a low endemic area for S. mansoni, and populations of the water-rat Nectomys squamipes act as reservoirs of the parasite. Schistosoma mansoni populations infecting humans and water-rats in Sumidouro present chronobiological variation in cercariae emergence (chronotype) and phenotypic characteristics between host species. Some studies have postulated that chronotype adjustment of S. mansoni populations to human (diurnal chronotype) and water-rat (nocturnal chronotype) hosts could result in prezygotic isolation. To test this hypothesis, we used the mitochondrial cytochrome c oxidase subunit 1 (MT-CO1) and microsatellite loci as genetic markers. We analysed the population structure of S. mansoni from the definitive host species and the spatial distance of the isolates between two endemic localities (PAM and ENC-SOL) in the municipality of Sumidouro, Brazil. We obtained 555 bp MT-CO1 sequences from 243 S. mansoni specimens, and seven microsatellite loci were genotyped for 158 S. mansoni specimens, including eggs and adult helminths from water-rat and eggs from human feces. Four haplotypes of the MT-CO1 gene were found, with haplotypes 1 and 2 shared by two studied localities and haplotypes 3 and 4 exclusive to the ENC-SOL locality. Haplotypes 1, 2 and 3 were shared between human and N. squamipes hosts. The population structure analysis revealed geographical structure and no structure associated with definitive host species, indicating that the populations of S. mansoni of different chronotypes are not isolated, with significant gene flow between them. In conclusion, our results confirm that wild rodents can contribute to the maintenance of the S. mansoni life cycle in Sumidouro and can be used as indicators of local transmission areas.