An etiology study on HFMD in Qingdao region during 2008-2009 was conducted. The virus RNA were isolated from throat swabs of HFMD,the EV, EV71 and CVA16 were detected by multiplex realtime RT-PCR. For those specimens with EV positive and both EV71 and CVA16 negative,a reverese transcription-seminested polymerase chain reaction (RT-snPCR) was perfomed to amplify part sequence of the VP1 gene for sabsequent analysis to identify the serotype. The results indicated that EV71 and CVA16 were the major pathogens of HFMD in Qingdao during 2008-2009. The proportion of EV71 was greater than CVA16 in either mild or serious HFMD cases. Sequence analysis showed that 5 non-EV71 and non-CVA16 serotypes (8 specimens) were obtained in 2008 including Coxsackievirus A5, A6, A10, A12 and Echovirus 9, which were well distributed. Three serotypes(13 specimens) were obtained in 2009 including Coxsackievirus A9, A12 and B2, of which CVA12 was of a big proportion (11/13). CVA12 became a new relatively major pathogen of HFMD in Qingdao during 2009.