Fibropapillomatosis (FP) is a tumor disease associated with a herpesvirus (Chelonid herpesvirus 5-ChHV5) that affects mainly green turtles globally. Understanding the epidemiology of FP has been hampered by lack of robust serological assays to monitor exposure to ChHV5. This is due in part to inability to efficiently culture the virus in vitro for neutralization assays. Here, we expressed two glycoproteins (FUS4 and FUS8) from ChHV5 using baculovirus. These proteins were immobilized on ELISA plates in their native form, and assayed for reactivity to two types of antibodies, the full length 7S and truncated 5.7S IgY. In Florida, turtles were uniformly seropositive regardless of tumor status. In contrast, in Hawaii, we detected strong antibody reactivity mainly from tumored animals with lower antibody response in non-tumored animals, including those from FP Enzootic areas. In Hawaii, turtles actively shedding ChHV5 were more seropositive than non-shedders. In trying to account for differences in serological responses to ChHV5 between Hawaii and Florida, we rejected cross reactivity of antibodies to other herpesviruses, differences in viral epitopes, or differences in procedure as likely explanations. Rather, behavioral differences between green turtles from Hawaii and Florida, respectively, have led to the emergence of biologically different viral strains. While Florida strains apparently spread independent of tumors, transmission of the Hawaiian subtype apparently relies heavily on tumor formation.