In the United States, cattle production is worth $50 billion. An important tick-borne disease affecting U.S. cattle is bovine anaplasmosis, costing around $300 million per year. Caused by the bacteria, Anaplasma marginale, anaplasmosis spreads via ticks, flies, or contaminated fomites. Worldwide, different Anaplasma species affect cattle (A. marginale), sheep and goats (A. ovis), and a broad host range: cattle, sheep, and humans (A. phagocytophilum). Currently, Anaplasma detection in cattle uses a USDA-approved cELISA, a serologic test that targets the highly conserved Anaplasma msp5 gene but does not distinguish among species. DNA-based molecular detection methods mainly involve nucleic acid extraction, amplification, and detection steps that are not suitable for field screening due to expensive laboratory equipment and trained personnel. A specific, sensitive, easy-to-use, and rapid detection method is needed to improve the accuracy of Anaplasma diagnosis. To address this need, four recombinase polymerase amplification (RPA) primers were designed using A. marginale, A. ovis, and A. phagocytophilum msp4 genes, and internal control based on GAPDH gene. To develop the RAD kit, infected blood was treated by Triton X-100 pre-lysis blood treatment then filtered by an elution independent collection device (EICD). Four discs of soluble central membrane (SCM) were punched from the EICD for use in the detection assay with each disc containing approximately 0.249 pg/µl of Anaplasma DNA. Nucleic acid recovered from in SCM was 60% - 70% of the total. A 20-minute RPA detection assay was developed at 37°C using 4 discs of EICD soluble membrane as template. Finally, RPA results were observed using either electrophoresis or lateral flow strips. The limit of detection of gel-based RPA was 0.1pg/µl for A. marginale, 1pg/µl for A. ovis and 1fg/µl for A. phagocytophilum; while, sensitivity of multiplex lateral flow RPA assays was 1 fg/µl detecting each Anaplasma spp. and GADPH gene. Both RPAs demonstrated no cross-reaction between the Anaplasma species. The applicability of lateral flow RPA technique was evaluated using 24 A. marginale positive serum samples. Twenty-two (92%) of the samples tested positive by RPA, while only one was positive by published endpoint PCR primers. The ideal samples for RPA assays should be blood because Anaplasma is inside the blood-red cells; however, lateral flow RPA also can also be used to test Anaplasma using serum samples. The RAD kit prototype will be practical for low-cost field use as well as a point-of-care diagnostic to identify and discriminate among the three Anaplasma species.