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Background: Nagana (African Animal Trypanosomiasis-AAT) and tick-borne diseases (TBDs) constrain livestock production in most parts of sub-Saharan Africa. To this realisation, Uganda government set up an African trypanosomiasis (AT) control unit, which among other activities generates national tsetse control priority maps using apparent tsetse density data. Such maps underestimate mechanically transmitted AAT and thus ought to be refined using actual AT prevalence data. We therefore set out to generate up-to-date cattle and donkey trypanosomiasis prevalence data as well as find out the constraints to livestock production in Karamoja region in a bid to re-define AT control priority in this region., Results: Livestock keepers and animal health workers indicated that TBDs and AAT were the most important livestock diseases in Karamoja region. The prevalence of Trypanosoma spp. in cattle and donkeys was 16.3% (95% CI: 12.4-21.1%) and 32.4% (95% CI; 20.2-47.6%) respectively. Trypanosoma vivax (12.1%) and Trypanosoma congolense savannah (29.6%) were the most prevalent Trypanosoma spp. in cattle and donkeys respectively. Majority of the cattle (85.7%) and more than half of the donkey (57.1%) herds were positive for Trypanosoma spp., Conclusions: African animal trypanosomiasis and TBDs are the most important constraints to livestock production in Karamoja region. In order to improve livestock production and hence Karamajong livelihoods, government of Uganda and her development partners will need to invest in livestock health programs particularly targeting tsetse and TBD control.