Bivalve hemocytes are involved in a variety of physiological and immunological functions. Circulating hemocytes in the hemolymph represent the main component of the internal self-defense system while hemocytes present in the extrapallial space (between the mantle and the shell) are actively involved in biomineralization and shell formation. This study focused on the characterization of hemocytes from different body fluids of the eastern oyster Crassostrea virginica. Hemocytes present in the hemolymph were compared to those contained in the extrapallial fluid. Hemocytes associated with the mucus layer covering pallial organs (mantle, gills, body wall) were also investigated because of their potential role as sentinel cells. Hemocytes were characterized using flow cytometry in conjunction with fluorescent epitope markers (clusters of differentiation, lectins) as well as functional assays (i.e. phagocytosis and reactive oxygen species -ROS). Compared with the hemolymph, there was a significantly greater percentage of granulocytes and agranulocytes among extrapallial and pallial hemocytes, respectively. Accounting for the different percentages of hemocyte sub-populations, significant differences in surface carbohydrate and clusters of differentiation signatures were also revealed between the different fluids. Most informative epitope markers included concanavalin A, peanut agglutinin, soybean agglutinin, CD11b and CD14. Functional assays revealed significant differences in phagocytic activity and ROS production between hemocytes from the extrapallial fluid and hemolymph; however, less robust differences were observed between hemolymph cells and hemocytes associated with the pallial mucus. Findings from this study suggest that there are markedly different hemocyte populations in the three body fluids. The role of peripheral cells, particularly those associated with the pallial mucus, requires further investigations.