A nested polymerase chain reaction (PCR) system was developed for the detection of Flexibacter maritimus from fish tissue. The total procedure for the diagnosis of marine flexibacteriosis, from the point of DNA extraction to the electrophoretic analysis, can be performed in <4 h. This was achieved by the combination of a short thermal cycling programme with a rapid DNA extraction procedure. The assay was extremely sensitive, capable of detecting as few as 75 cfu mg−1 fish tissue. The accuracy of the nested PCR was confirmed under field conditions using tissue samples recovered during 1993–2002 from fish suffering marine flexibacteriosis. The nested PCR method proved to be efficient for the rapid and sensitive detection of F. maritimus from fish tissues and can be used for routine diagnosis of the disease caused by this pathogen.