Our overall aim is to develop epitope-based assays for accurate differential diagnosis of B virus zoonotic infections in humans. Antibodies to cross-reacting epitopes on human-simplexviruses continue to confound the interpretation of current assays where abundant antibodies exist from previous infections with HSV types 1 and 2. To find B virus-specific epitopes we cloned ten monoclonal antibodies (mAbs) from the hybridomas we produced. Our unique collection of rare human sera from symptomatic and asymptomatic patients infected with B virus was key to the evaluation and identification of the mAbs as reagents in competition ELISAs (mAb-CE). The analysis of the ten mAbs revealed that the target proteins for six mAbs was glycoprotein B of which two are reactive to simian simplexviruses and not to human simplexviruses. Two mAbs reacted specifically with B virus glycoprotein D, and two other mAbs were specific to VP13/14 and gE-gI complex respectively. The mAbs specific to VP13/14 and gE-gI are strain specific reacting with B virus isolates from rhesus and Japanese macaques and not with isolates from cynomolgus and pigtail macaques. The mAb-CE revealed that a high proportion of naturally B virus infected rhesus macaques and two symptomatic humans possess antibodies to epitopes of VP13/14 protein and on the gE-gI complex. The majority of sera from B virus infected macaques and simplexvirus-infected humans competed with the less specific mAbs. These experiments produced a novel panel of mAbs that enabled B virus strain identification and confirmation of B virus infected macaques by the mAb-CE. For human sera the mAb-CE could be used only for selected cases due to the selective B virus strain-specificity of the mAbs against VP13/14 and gE/gI. To fully accomplish our aim to provide reagents for unequivocal differential diagnosis of zoonotic B virus infections, additional mAbs with a broader range of specificities is critical.