Ehrlichia ruminantium is a tick-borne pathogen transmitted by ticks in the genus Amblyomma. This bacterial pathogen causes heartwater, a fatal disease affecting domestic and wild ruminants in sub-Saharan Africa. The prevalence of heartwater in western Kenya is not well documented. In this study, reverse line blot (RLB) hybridization and quantitative real-time PCR (qPCR) assays were used to detect E. ruminantium DNA in 545 blood samples collected from calves from twenty sublocations distributed across five agro-ecological zones of western Kenya. Ehrlichia ruminantium DNA was detected in 1.10% and 0.92% of the samples using RLB and qPCR, respectively. There were discrepancies in the detection of E. ruminantium by the RLB and the qPCR. Five samples were positive with the qPCR while six were positive with the RLB, but only three of the samples were positive by both tests. The occurrence of E. ruminantium in western Kenya appears to be low, but this might be attributed to the inability of the tests used to detect E. ruminantium carriers. The most prevalent haemoparasites detected by the RLB in the Ehrlichia/Anaplasma group were Anaplasma (formerly Ehrlichia) sp. Omatjenne and Anaplasma bovis at 37.98% each, while Theileria mutans (66.61%) was the most prevalent in the Theileria/Babesia group. In addition, a nested p104 PCR was used to detect Theileria parva in a subset of 86 of the samples; T. parva was detected in 32.56% (28/86) of these samples. The RLB detected T. parva in 27.91% (24/86) of the same sample subset, but only 17 were positive by both tests. The molecular tests used in this study suggest that, of the pathogenic haemoparasites known to cause disease in Kenya, T. parva occurs the most commonly in western Kenya, while E. ruminantium, A. marginale, B. bigemina and B. bovis are less frequently detected.