Objective To evaluate the application value of PCR-sequencing in clinical detection of hantavirus in rodents. Methods Based on 7 subtypes and 24 strains of representative hantavirus strains downloaded from Genbank, the virus S gene fragments were used for primer design and neighbor joining method was applied for phylogenetic analysis. Thereafter, we identified hantavirus strains isolated from wild rodents in recent years in Zhejiang Province by this method. Results The 24 analyzed strains were divided into 5 regions in the phylogenetic tree. Four of them with topology structure were more stable. Eleven strains of the virus were amplified by PCR and sequenced, and the results showed that the primers were with high sensitivity and specificity. Three HTN strains and 1 strain of serotype SEO were distinguished from 9 strains of unknown strains isolated in Zhejiang Province. We also found that 5 strains of hantavirus belonging to two unknown serotypes. Discussion Our results suggest that the PCR-sequencing method proposed in this study can be used for clinical detection of hantavirus.