The development of diagnostic assays more sensitive and specific than traditional histological techniques is important for the management of bonamiasis in flat oysters Ostrea edulis. A specific polymerase chain reaction (PCR) protocol was developed for the detection of very small amounts of Bonamia ostreae (Pichot et al. 1980) ribosomal DNA (rDNA) in bulk DNA from oyster gill and hemolymph. The presence of a 760 bp PCR amplification product corresponded with B. ostreae infections determined cytologically in 185 oysters from Ireland, Spain, and the USA. All (100%) 'heavily' and 'moderately' infected oysters, 86.7 % of the 'lightly' infected oysters, and 66.7 % of the 'scarcely' infected oysters were confirmed to be infected using the PCR. In addition, 37.9% of the oysters in which B. ostreae was not detected using cytology were positive using the PCR. Sampling error and the subjectivity of cytological diagnoses are the likely sources of disagreement between diagnostic methods in oysters with very light infections. The PCR assay developed here is more sensitive and less ambiguous than standard histological and cytological techniques. Phylogenetic analysis of DNA sequence data confirmed B. ostreae to be a member of the Haplosporidia.