A total of 105 hybridomas secreting anti-Japanese encephalitis (JE) virus monoclonal antibodies (mAbs) were generated from six fusions against four strains of JE virus: wild-type strains SA14 and G8924 and live attenuated vaccines SA14-5-3 and SA14-14-2 (PDK-9). Most of the mAbs (87%) elicited haemagglutination inhibition activity while only a minority (24%) elicited neutralization. None of the mAbs prepared against SA14-5-3, parent of SA14-14-2, elicited neutralization while the only mAbs prepared against SA14-14-2 that elicited neutralization recognized flavivirus cross-reactive epitopes. In comparison, mAbs raised against wild-type strains showed that a spectrum of epitopes with different specificities, including JE type-specific epitopes, elicited neutralizing activity. Two mAbs, prepared against SA14-5-3 virus, were found to be vaccine-specific and five, prepared against strains SA14 and G8924, were wild-type-specific.