Polymerase chain reaction (PCR) assay and other techniques were applied to differentiate Hantavirus strains isolated from different animal hosts and geographic regions in China. Two groups of related strains, Hantaan and Seoul, have been classified by cross-neutralization, radioimmunoprecipitation (RIP), and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) assays. The molecular weights of glycoprotein 1 (G1) of Hantaan and Seoul viruses were 72k and 80k, whereas those of the nucleocapsid (N) and glycoprotein 2 (G2) remained the same, respectively. The PCR assay was used to differentiate these isolates using synthetic oligonucleotide primers selected from various regions of the M genome of 76118 and R22 strains. 76118-specific primers amplified only the RNAs extracted from Hantaan strains while R22-specific primers, the RNAs from Seoul strains. The PCR results for classification are consistent with those obtained by cross-neutralization, RIP and SDS-PAGE assays.