KI polyomavirus, which was firstly discovered in 2007, is a new human polyomavirus belonging to Polyomaviridae and containing circular double-strand genomic DNA. This study was based on identification assay of KI polyomavirus reported. Total 2293 clinical sputum specimens from children under 3-years-old were collected and screened from Wenzhou Medical College affiliated Wenling Hospital, Zhejiang Province. A KI polyomavirus was detected and identified, the positive rate was 0.04%. The sequences of PCR products was identical to that of the viral capsid protein (VP1) gene derived from KI polyomavirus. The results strongly suggested that the KI polyomavirus was found firstly in Chinese children with acute lower respiratory tract infections from Zhejiang region. This study provided new information for further investigation of etiopathogenisis and diagnosis in children with lower respiratory tract infections.