Species of Hematodinium are endoparasitic dinoflagellates of crustaceans. Certain stages of the parasites can be very difficult to detect in the hemolymph of their hosts, because the trophic stages resemble hemocytes, and they can occur at relatively low densities, making diagnosis by microscopy difficult. We developed a polymerase chain reaction (PCR) assay to detect the Hematodinium sp. infecting the blue crab, Callinectes sapidus, based on the amplification of the parasite's first internal transcribed spacer region (ITS1) of the ribosomal RNA (rRNA) gene complex. The PCR assay was combined with a restriction endonucleases digestion (Bsg I) of the amplification products to differentiate between different forms of Hematodinium from different hosts. The assay had a limit of detection equivalent to 0.3 parasites per 100-μL hemolymph. In addition, two oligonucleotide DNA probes were designed to target the 18S rRNA gene sequence of the parasite, facilitating detection in situ in crustacean tissues. These probes appear to target several, if not all species within the genus, because they labeled all isolates of Hematodinium tested in this study, whereas they were not hybridizing to other parasite species. The PCR-RFLP assay will be invaluable for future studies investigating parasite prevalence, the existence of secondary hosts or environmental reservoirs, and modes of transmission, whereas the DNA probes will be useful for confirming and localizing Hematodinium parasites in crustacean tissues.