A cross-sectional study was performed 200 blood samples and 314 tick samples in El Huda and El Nuhud animals production research stations, Sudan, in May (summer) and December (winter) in 2016, to determine the prevalence of
Theileria lestoquardi
in sheep and the potential risk factors associated with the infection. A total of 200 blood samples and 314 tick samples were collected from El Huda (n = 103 blood, 97 tick) and El Nuhud (n = 97 blood, 217 tick) stations. Data on the risk factors, such as age, sex, ecotype of sheep, body condition score and seasons were recorded. The overall prevalence of
Theileria lestoquardi
was 13% (26/200) using PCR. A significant variation in the prevalence of
Theileria lestoquardi
was observed between the stations and the ecotype of sheep (
p
≤ 0.05), whereas the highest prevalence was recorded in El-Huda station (19.4%) as well as in Shugor (22.8%). Other risk factors, like age, sex, body condition, and seasons were not found to be significantly associated with infection. However, the highest prevalence rate was recorded in old animals (21.6%) than the other, in males (17.9%) than females (12.2%), in animals with poor body condition (26.1%) than the other, and in winter (16%) than summer (10%). Four tick species i.e.
Rhipicephalus evertsi evertsi
(63.1%),
Hyalomma anatolicum
(13.8%),
Hyalomma dromedarii
(8.8%), and
Hyalomma impeltatum
(14.3%) were recorded in El Nuhud station. While in El Huda station, only
Rhipicephalus evertsi evertsi
(79.4%),
Hyalomma anatolicum
(20.6%) were recorded. This study revealed that 13% of sheep were suffering from
Theileria lestoquardi
which is a considerable number at the stations. Therefore, further epidemiological investigations on disease throughout the year are required in order to set a well-coordinated control program.