Dinoflagellate parasites in the Hematodinium genus have emerged as important pathogens of economically important crustaceans worldwide, causing significant economic losses to fisheries and aquaculture. An understanding of the routes of infection in blue crab (Callinectes sapidus) populations would be facilitated by an improved knowledge of environmental reservoirs. A previously used PCR assay, based on small subunit rRNA sequences, lacked the specificity needed for Hematodinium perezi detection of environmental samples. Therefore a quantitative PCR assay based on the internal transcribed spacer 2 (ITS2) region of H. perezi rRNA genes was developed, validated, and applied to examine temporal and spatial incidences of environmental reservoirs in Delmarva coastal bays. H. perezi was detected in sediment and water in several Delmarva coastal bays, as well as the host, C. sapidus. Results suggest the existence of localized sediment reservoirs in areas where hydrological and geophysical features allow for the formation of cell deposits.