The utility of a functional immune assay for smallmouth bass (Micropterus dolomieu) lymphocyte mitogenesis was evaluated. Wild populations in the Potomac River have faced disease and mortality with immunosuppression from exposure to chemical contaminants a suspected component. However, a validated set of immune parameters to screen for immunosuppression in wild fish populations is not available. Prior to use in ecotoxicology studies, ancillary factors influencing the mitogenic response need to be understood. The assay was field-tested with fish collected from three sites in West Virginia as part of health assessments occurring in spring (pre-spawn; April–May) and fall (recrudescence; October–November). Anterior kidney leukocytes were exposed to lipopolysaccharide (LPS) from E.coli O111:B4 or mitogen-free media and proliferation was measured using imaging flow cytometry with advanced machine learning to distinguish lymphocytes. An anti-smallmouth bass IgM monoclonal antibody was used to identify IgM+ lymphocytes. Lymphocyte mitogenesis, or proliferative responses, varied by site and season and positively and negatively correlated with factors such as sex, age, tissue parasites, and macrophage aggregates. Background proliferation of IgM− lymphocytes was negatively correlated to LPS-induced proliferation in both seasons at all sites, but only in spring for IgM+ lymphocytes. The results demonstrate that many factors, in addition to chemical contaminants, may influence lymphocyte proliferation.