BACKGROUND The present study, conducted in Zambia's Luangwa valley where both animal African trypanosomiasis (AAT) and human African trypanosomiasis (HAT) are endemic, combined the use of microscopy and molecular techniques to determine the presence of trypanosome species in cattle, goats and tsetse flies. METHODS This study was conducted between 2008 and 2010 in Petauke, Chama and Isoka districts, north-eastern Zambia. A total of 243 cattle, 36 goats and 546 tsetse flies, were examined for presence of trypanosome species using microscopy, PCR and loop-mediated isothermal amplification (LAMP). RESULTS There was poor agreement among the test methods used for detection of trypanosomes species in animal blood and tsetse flies. Trypanosomes were observed in 6.1 % (95 % CI: 3.3-8.9 %) of the animals sampled by microscopy, 7.5 % (95 % CI: 4.4-10.6 %) by PCR and 18.6 % (95 % CI: 13.6-23.6 %) by PFR-LAMP. PFR-LAMP was more sensitive for detecting Trypanozoon than KIN-PCR. The highest occurrence of AAT was recorded in cattle from Petauke (58.7 %, 95 % CI: 44.7-72.7 %) while the lowest was from Isoka (5.4 %, 95 % CI: 0.8-10.0 %). Infection of both cattle and goats with Trypanosoma congolense and T. vivax was associated with clinical AAT. CONCLUSIONS When selecting molecular techniques for AAT surveillance in endemic regions, the KIN-PCR and species-specific PCR may be recommended for screening animal or tsetse fly samples for T. congolense and T. vivax, respectively. On the other hand, species-specific PCR and/or LAMP might be of greater value in the screening of animal and human body fluids as well as tsetse fly samples for Trypanozoon.