Background Ticks and tick-borne diseases (TTBDs) are a significant threat to livestock production in sub-Saharan Africa. Transhumance pastoralism practiced in Karamoja region and other factors like cattle trade, communal grazing and the presence of wildlife predispose cattle to TTBDs. Tick species abundance and distribution data can be used as a tool for early disease diagnosis and inform tick control strategies. However, these data for north-eastern Uganda are currently limited; previous surveys were relatively localized and targeted fewer cattle kraals and numbers.Methods We randomly collected tick specimens from 1,534 cattle spread across Karamoja region in both the peak month of the rainy (May) and the dry (February) seasons. The ticks were identified using morpho-taxonomic keys and the tick species identities confirmed by the 16S rRNA gene sequencing and phylogenetic analysis.Results A collection of 18,637 ticks was examined and 15 tick species from three genera (10 Rhipicephalus; 3 Amblyomma; 2 Hyaloma species) were identified. Rhipicephalus appendiculatus was the most dominant (37.9%) tick specie, followed by Amblyomma variegatum (32.3%); A. lepidum (17.3%); R. evertsi evertsi (7.8%); and R. decoloratus (1.4%). Eight of these tick species were ubiquitous in the study districts while six were found in isolated areas. The peak month of the dry season collection was associated with a higher proportion of tick-infested cattle (91%) compared to the peak month of the rainy season (89.8%); a difference that we did not find statistically significant (χ2 = 0.5077, n = 1385, p = 0.476). The overall cattle infestation rate was mainly dominated by 5 tick species namely: A. variegatum (55%), R. appendiculatus (53%), A. lepidum (41%), R. Evertsi (22%), and R. Decoloratus (8%). Napak and Amudat districts registered the highest (95.4% and 94.9%) and lowest (80.9% and 80.7%) cattle tick infestation rates during the peak months of the rainy and dry seasons respectively. Rhipicephalus microplus was confirmed in Amudat, Kaabong and Napak districts.Conclusion This study demonstrates high cattle tick infestation rates by a battery of tick species in Karamoja region. We identified both R. microplus and R. decoloratus which indicates that R. microplus has recently been introduced in this region. This calls for effective tick control responses to prevent further spread of this invasive cattle tick specie.