We genetically analysed haematozoa parasites (Protozoa) isolated from nestling eagle owls (Bubo bubo) in Toledo province, Central Spain. A total of 206 nestlings from 74 nests were screened for parasites of the genera Leucocytozoon, Plasmodium, and Haemoproteus using a very efficient polymerase chain reaction (PCR) approach that amplifies a partial segment of the mitochondrial cytochrome b gene of these parasites. PCR-based detection and sequence analyses revealed a unique lineage of Leucocytozoon (EO1) parasitizing nestling eagle owls. Ocular examination of blood smears identified these parasites as Leucocytozoon ziemanni, the only species deemed valid in owls based on morphology. Preliminary phylogenetic analyses using homologous published sequences isolated from other owl species suggest that L. ziemanni constitutes a monophyletic clade that may be composed by a complex of genetically differentiated cryptic species.