The control of African trypanosomiasis (AT) in Eastern and Southern Africa, including Zambia, faces huge challenges due to the involvement of wild and domestic animal reservoirs. Free-roaming dogs in wildlife-populated and tsetse-infested villages of Zambia’s Mambwe district are exposed to infectious tsetse bites. Consuming fresh raw game meat and bones further exacerbates their risk of contracting AT. We focus on the reservoir role of such dogs in maintaining and transmitting diverse species of trypanosomes that are infective to humans and livestock in Zambia’s Mambwe district. A cohort of 162 dogs was enrolled for follow-up at 3 different time points from June to December 2018 in selected villages of Malama, Mnkhanya, and Nsefu chiefdoms of Mambwe district, eastern Zambia. Blood and serum were screened for AT by microscopy, GM6 ELISA, PCR (ITS1 and SRA), and Sanger sequencing. Out of the 162 dogs in the cohort, 40 were lost to follow-up and only 122 remained traceable at the end of the study. GM6 ELISA detected Trypanosoma antibodies in 121 dogs (74.7%) and ITS1-PCR detected DNA involving single and mixed infections of T. congolense, T. brucei, and suspected T. simiae or T. godfreyi in 115 dogs (70.9%). The human-infective T. b. rhodesiense was detected by SRA PCR in 67 dogs (41.4%), and some sequence data that support the findings of this study have been deposited in the GenBank under accession numbers OL961811, OL961812, and OL961813. Our study demonstrates that the Trypanosoma reservoir community in Zambia is wider than was thought and includes domesticated dogs. As dogs are active carriers of human and livestock-infective trypanosomes, they pose a risk of transmitting AT in endemic villages of Mambwe district as they are neglected and left untreated. To fully bring AT under control, countries such as Zambia where the role of animal reservoirs is important, should not limit their prevention and treatment efforts to livestock (especially cattle) but also include dogs that play an integral part in most rural communities.