Abstract： Hand,foot and mouth disease(HFMD)is a major public health problem among children in the Asia-Pacific region.The optimal specimen for HFMD virological diagnosis remains unclear.Enterovirus A71(EV-A71)neutralizing antibody titres detected in paired sera were considered the reference standard for calculating the sensitivity,specificity,positive and negative predictive value of throat swabs,rectal swabs,stool,blood samples and cere-brospinal fluid(CSF)by RT-PCR or ELISA assay.In this study,clinical samples from 276 HFMD patients were collected for analysing the sensitivity of different kind of specimens.Our results showed that stool had the highest sensitivity(88％,95％CI:74％-96％)and agreement with the reference standard(91％).The order of diagnostic yield for EV-A71 infection was stool sample≥rectal swab＞throat swab＞blood sample＞CSF sample,and using a combination of clinical samples improved sensitivity for enterovirus detection.The sensitivity of ELISA for IgM antibody detection in sterile-site specimens was significantly higher than that of RT-PCR(serum/plasma:62％vs.2％,CSF:47％vs.0％)(P＜0.002).In conclusion,our results suggest that stool has the highest diagnostic yield for EV-A71-infected HFMD.If stool is unavailable,rectal swabs can be collected to achieve a similar diagnostic yield.Otherwise,throat swabs may be useful in detecting positive samples.Although IgM in blood or CSF is diagnos-tically accurate,it lacks sensitivity,missing 40％-50％of cases.The higher proportion of severe cases and shorter interval between onset and sampling contributed to the increase in congruency between clinical testing and the serological reference standard.