The recent resurgence of mumps epidemics in many countries with ongoing vaccination programs along with evidence of antigenic diversity among mumps virus strains have recently challenged the assumption that mumps virus is serologically monotypic. To address this controversy, we sought to identify two mumps virus strains that would best represent different serotypes, should multiple serotypes exist, and assess the ability of human sera to neutralize both strains. The virus strains, Enders and Lo1, were selected based upon a phylogenetic analysis of the major target of neutralizing antibody, the viral hemagglutinin-neuraminidase (HN) protein, along with data reported by others indicating that (1) these viruses are antigenically distinct and (2) genotypically similar strains have been implicated in cases of reinfection. Our results show that of sera capable of neutralizing one of the virus strains, 90% could neutralize the other, although significant differences in neutralization titers were noted. Though the latter confirms the existence of inter-strain antigenic variability, the fact that few sera were unable to neutralize both virus strains argues against the presence of multiple serotypes. Of those sera incapable of co-neutralization, all but one had low neutralization titers (1:8), suggesting that individuals possessing low levels of neutralizing antibody may be at risk for breakthrough infections, thereby providing an explanation for cases of infection in previously infected or vaccinated individuals.