Background: The discovery of an unexpected genetic sequence raised doubts about the specificity of a primer pair targeting Babesia spp. and Theileria spp. This study aimed to check the specificity of this primer pair. Methods: Conventional end-point PCR and real-time PCR protocols using primers targeting the 18S rRNA gene V4 hypervariable region of Babesia spp. and Theileria spp. were tested for potential cross-genera amplification using DNA from a palette of parasitic protists and pathogenic bacteria as a template. These investigations took place at the Ludwig Maximilian University of Munich (Germany) in 2010 as part of the EDEN project. Results: Successful amplification was obtained with DNA from five apicomplexan genera: Babesia, Theileria, Hepa tozoon, Toxoplasma, and Hammondia. No amplicons were obtained when DNA from Leishmania infantum or bacte ria within the genera Borrelia, Leptospira or Anaplasma was used as a template. Conclusion: This cross-genera amplification ability is useful for the quick exclusion of many parasite species from PCR negative diagnostic samples. Accurate species identification from PCR positive samples requires genetic se quencing of the amplicon.