A simple and sensitive method was developed for the differentiation of the Hoshino vaccine strain from wild strains with a restriction fragment length polymorphism (RFLP) analysis in the part of hemagglutinin-neuraminidase (HN) gene. The virus genome was amplified by using a reverse transcriptase-polymerase chain reaction (RT-PCR) directly from clinical samples. The PCR product of the Hoshino vaccine strain was cleaved into 2 fragments after digestion with Sca I and Afl II. All wild strains showed 2 RFLP profiles, A and B, different from that of vaccine strain. Wild A strains were cut into 2 fragments after digestion with Sca I only, while wild B strains were cleaved neither with Sca I nor Afl II. This molecular approach provides an effective method for differentiation of the Hoshino vaccine strain from wild strains of mumps virus in patients after vaccination.