Bonamia ostreae is the causative agent of the disease bonamiosis, which has depleted stocks of the European flat oyster, Ostrea edulis, since the parasite was introduced into Europe in the late 1970s. Accurate, sensitive and rapid diagnostic techniques are essential in the detection of B. ostreae and in the implementation of oyster transfer restrictions governed under the EU Directive (2002/878/EC) and OIE, 2006, and by August 2008 the EU Directive (2006/88/EC). The aim of this study was to compare the different diagnostic techniques prescribed to detect B. ostreae: heart smears, histology, Polymerase chain reaction (PCR) and in situ hybridisation (ISH): for their reliability and sensitivity in detecting the parasite. This is the first study to compare ISH to other diagnostic methods. In many studies and monitoring programs due to time and financial constraints, screening of animals may involve one or a combination of techniques with, in some instances, only positive samples being further confirmed by the use of additional techniques. In this study, 713 oysters were first screened by heart smear technique and PCR for the presence of B. ostreae. All positives, derived from one or both of these methods, were further screened using histology and ISH. This allowed a comparison of the four techniques and investigated their reliability as a further confirmatory test for B. ostreae. Sensitivity of PCR, histology and ISH was compared relative to intensity of B. ostreae infection as determined by screening of heart smears. When all positive results were compared, the results showed that each diagnostic technique failed to detect some infections of B. ostreae. 13% of class 3 infections were detected by only one diagnostic technique. Overall heart smear examination was the most sensitive individual technique, while ISH was the least sensitive. A greater sensitivity of detection was obtained when results of heart smear and PCR screening were combined. (C) 2008 Elsevier B.V. All rights reserved..