Perkinsus marinus is a major pathogen of the eastern oyster Crassostrea virginica. Elucidating the spatial distribution of genetic variation within the species is critical to an examination of potential virulence differences among strains and to understanding the implications of transferring infected oyster stocks. The genetic similarity of P. marinus in vitro cultures and their clonal composition was examined using isolates from individual oysters collected from 1991 through 1999 along the Atlantic and Gulf of Mexico coasts of the USA. P. marinus has been endemic to the Atlantic coast from the Virginia portion of Chesapeake Bay southward and into the Gulf of Mexico since its initial description in 1949, and over the last 10-15 years there has been a range expansion of this parasite into the Maryland portion of Chesapeake Bay and northward along the Atlantic coast from New Jersey to Maine. DNA purified from 76 primary (parental) cultures and 86 clonal cultures derived from the parental isolates was examined at eight polymorphic loci by restriction fragment length polymorphism analysis. Comparison of clonal and parental culture genotypes supported previous observations that isolate cultures initiated from a single oyster can be polyclonal, providing evidence that an individual oyster can be infected with multiple strains. Allelic and genotypic frequencies differed significantly among three regions of the USA; the Northeast Atlantic (Maine to Maryland), the Southeast Atlantic (Virginia to Florida's east coast), and the Gulf of Mexico (Florida's west coast to Texas). Overall, 12 different composite genotypes were detected in this study, with > 88% of isolates possessing one of three predominant genotypes. One of the major composite genotypes was unique to Gulf coast isolates, while the genetic strain of P. marinus detected most frequently in oysters from the Northeast was not found in Gulf coast oysters. Perkinsus marinus is ubiquitous along the Atlantic and Gulf coasts, but different regions possess unique assemblages of genetic strains. Apparently, the historically common practice of oyster transplantation between regions has not significantly altered the population genetic structure at the regional level. The data suggest a founder event in the Northeast region, corresponding to the recent range expansion. Previously documented differences in virulence are consistent with genotypic differences, indicating the potential to identify strain virulence with molecular genetic tools.