Cultures of four strains of the dinoflagellate Gymnodinium aureolum (Hulburt) G. Hansen were established from the Elizabeth River, a tidal tributary of the Chesapeake Bay, USA. Light microscopy, scanning electron microscopy, nuclear-encoded large sub-unit rDNA sequencing, and culturing observations were conducted to further characterize this species. Observations of morphology included: a multiple structured apical groove; a peduncle located between the emerging points of the two flagella; pentagonal and hexagonal vesicles on the amphiesma; production and germination of resting cysts; variation in the location of the nucleus within the center of the cell; a longitudinal ventral concavity; and considerable variation in cell width/length and overall cell size. A fish bioassay using juvenile sheepshead minnows detected no ichthyotoxicity from any of the strains over a 48-h period. Molecular analysis confirmed the dinoflagellate was conspecific with G. aureolum strains from around the world, and formed a cluster along with several other Gymnodinium species. Morphological evidence suggests that further research is necessary to examine the relationship between G. aureolum and a possibly closely related species Gymnodinium maguelonnense.