| Summary: | Ticks transmit many pathogens with public health and veterinary importance. Despite the wide distribution of tick-borne pathogens in Sudan, the information on the tick–pathogen relationship needs to be updated, particularly using modern molecular techniques. This cross-sectional study, conducted between September and November 2019, used morphology, PCR, and sequencing to confirm the identity of adult cattle ticks (male and female; n = 536) from Khartoum State (n = 417) and East Darfur State (n = 119). Moreover, the presence of <i>Theileria annulata</i>, <i>Babesia bigemina</i>, <i>B. bovis</i>, <i>Anaplasma marginale</i>, and <i>Ehrlichia ruminantium</i> was detected and confirmed in each tick using species-specific PCR or nested PCR and sequencing. The most economically important tick genera, <i>Rhipicephalus, Hyalomma</i>, and <i>Amblyomma</i>, were prevalent in the study area, and 13 different tick species were identified. The most prevalent tick species were <i>Rhipicephalus</i><i>evertsi evertsi</i> (34.3%) and <i>Hyalomma anatolicum</i> (57.3%) in Khartoum State, and <i>Rhipicephalus annulatus</i> (27%), <i>Rhipicephalus decoloratus</i> (25%), and <i>Hyalomma rufipes</i> (29%) in East Darfur State. We detected all five pathogens in both states. To the best of our knowledge, this is the first study to report the presence of <i>E. ruminantium</i>, its vector <i>Amblyomma variegatum</i>, and <i>B. bovis</i> in Khartoum State. Further, this is the first report on most tick and pathogen species identified in East Darfur State. Our findings indicate the migration of some tick and pathogen species beyond their distribution areas in the country, and this consideration is necessary to develop future control strategies.
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