Panulirus argus Virus 1 (PaV1) is the first virus known to be pathogenic to a wild lobster. It infects the Caribbean spiny lobster P. argus from the Florida Keys, and has a predilection for juveniles. The monitoring of the virus in wild populations and study of its behavior in the laboratory require the development of reliable diagnostic tools. A sensitive and specific fluorescence in situ hybridization (FISH) assay was developed for detection of PaV1. The lower detection limit using a 110 bp DNA probe in a dot-blot hybridization for PaV1 DNA was 10 pg of cloned template PaV1 DNA and 10 ng of genomic DNA extracted from the hemolymph of diseased spiny lobster. The fluorescein (FITC)-labeled probe specifically hybridized to PaV1-infected cells in the hepatopancreas, hindgut, gills, heart, foregut, and nerve tissues. FITC staining was observed around the inner periphery of the nuclear membrane, with lighter staining in a more dispersed pattern within the nucleus. The probe did not hybridize with host tissues of uninfected spiny lobsters, nor did it cross-react with 4 other virus samples tested. This assay will facilitate our understanding of the pathogenesis of the viral disease and help in monitoring efforts directed at determining the prevalence of PaV1 in juvenile nurseries for this lobster.