Fish kills and possible adverse human health effects have been attributed to Pfiesteria piscicida and other toxin producing estuarine heterotrophic dinoflagellates in US mid-Atlantic estuarine waters. Identification of these organisms in field samples (and, for that matter, in dinoflagellate culture) is problematic, and currently requires screening by light microscopy for the presence of \'pfiesteria-like-organisms\'; cultivation in the presence of fish as prey species (to confirm toxicity); and culture amplification to permit scanning electron microscopy (for definitive morphological identification based on thecal plate configuration). Genetic characterization of these organisms has been hampered by the lack of an axenic culture system. We adopted a molecular ecological approach utilizing PCR primers with Dinophyte specificity targeted to 18s rRNA gene sequences. These primers, whose specificity was demonstrated against an array of eukaryotic algal and protozoal species, were used for specific amplification of dinoflagellate gene sequences among complex genomic pools (non-axenic algal/dinoflagellate cultures and field samples of estuarine waters). Utilizing DNA extracted from active fish-kill bioassay cultures and environmental samples collected during fish kill events, dinoflagellate sequence diversity within amplified dinoflagellate 18s gene sequences was assessed by heteroduplex mobility assay (HMA), single stranded conformational polymorphism (SSCP), and sequencing. Use of the HMA was particularly helpful in identifying mixed dinoflagellate cultures amongst panels previously believed to be unialgal. A particular HMA pattern was consistently identified among cultures (nine) identified both by toxicity and scanning electron microscopy as Pfiesteria piscicida. PCR products with this signature pattern were cloned and sequenced, leading next to identification of the full-length 18s DNA sequence of Pfiesteria piscicida. P. piscicida specific PCR primers were then developed, and their specificity confirmed against panels of known dinoflagellate species. A real-time PCR assay (TaqmanR) has been developed to permit rapid and quantitative identification of the organism in estuarine waters.