Previous studies using ELISA and virus neutralization test (VNT) have proved the presence of Murine gammaherpesvirus 68 (MHV-68) serum antibodies in sera of laboratory staff working with MHV-68, as well as in the general population. In this study, we investigated the incidence of serum antibodies to MHV-68 and to human herpesviruses presumably antigenically similar to MHV-68, as Herpes simplex virus 1 (HSV-1), Human cytomegalovirus (HCMV), and Epstein-Barr virus (EBV), in general population using ELISA, VNT, and immunofluorescence assay (IFA). We also searched for the possible detection of false-positive reaction of MHV-68 antibodies due to cross-reactions between MHV-68 and the antibodies to the herpesviruses mentioned above. We found 16% of positive sera for MHV-68 antibodies by ELISA (titers of 1,600-102,400) and 4.5% by VNT and IFA (titers of 8-32). Tested human sera, either positive or negative for MHV-68 antibodies, were positive for antibodies to HSV-1, HCMV, and EBV (71/69%, 69/65%, and 66/49%, respectively). We concluded that the false-positivity of the sera for MHV-68 antibodies detected by ELISA was due to the nonspecific cross-reactions with antibodies to antigenically similar EBV.