Spiroplasma eriocheiris is the first spiroplasma strain known to be pathogenic to freshwater crustaceans.It has caused considerable economic losses both in the freshwater crayfish Procambarus clarkii (Girard) and some other crustaceans.The monitoring of the pathogen in crustacean populations and study of its behavior in the laboratory require the development of reliable diagnostic tools.In this article, we improved microscopic identification of S.eriocheiris by combining in situ hybridization with specific fluorescently labeled oligonucleotide probes.The established fluorescence in situ hybridization (FISH) allowed simultaneous visualization, identification and localization of S.eriocheiris in the tissues of diseased crayfish P.clarkii and exhibited low background autofluorescence and ideal signal-to-noise ratio.With the advantages of better tissue penetration, potentially more specific and stable, we designed three species-specific oligonucleotide probes utilizing the sequences of 16S-23S rRNA intergenic spacer regions (ISRs) of S.eriocheiris.Positive hybridization signals were visuahzed in hemocytes and connective tissues of hepatopancreas, cardiac muscle and gill from diseased crayfish.This unique distribution pattern matched the pathological changes when diagnosed by H&E staining and indicated that S.eriocheiris probably spread throughout the tissues in P.clarkii by hemokinesis.This assay will facilitate our understanding of the pathogenesis of S.eriocheiris and enhance the early diagnosis of the novel pathogen.