<p>Bovine Babesiosis is one of the tick-borne diseases (TBD) caused by <i>Babesia spp</i> and is transmitted by tick vectors of the genus <i>Rhipicephalus microplus</i>. The disease causes significant economic loses to livestock farmers and it has been reported to be endemic in many countries of East African region including Burundi. A recent study conducted in Burundi showed that the vectors of the disease are present in different agro-ecological zones. A cross-sectional study was conducted in the Imbo region to assess the prevalence and risk factors associated with bovine babesiosis. A total of 400 blood samples drawn from cattle were collected from either jugular or coccinea vein. Nested Polymerase Chain Reaction (nPCR) assay based on rhoptry associated protein-1a (<i>Rap-1a)</i>, and spherical body protein-4 (<i>SBP-4)</i> was used for pathogen detection and the resulting data was used to determine the risk factors using the R statistical software. Gene sequences and the genetic characterization were determined for selected positive samples. Out of a total of 400 samples, 5.50% (3.3–7.8, 95% CI) were found to be infected with <i>Babesia spp</i> where 4.25% (2.3–6.2, 95% CI) and 1.25% (0.2–2.3, 95% CI) were infected with <i>B. bigemina</i> and <i>B. bovis</i> respectively. Age, sex, tick infestation, grazing system, and origin (commune) were found to be significantly associated with bovine babesiosis (<i>P</i> value &lt; 0.05). The sequence analysis revealed that different genotypes of <i>B. bigemina</i> and <i>B. bovis</i> are present in the Imbo region and these results will help the competent authorities to design an effective strategy for the control of tick-borne diseases to reduce the economic losses causes by the diseases.</p>