Three armored RNAs (virus-like particles [VLPs]) containing target sequences from enterovirus 71 (EV71) and coxsackievirus A16 (CA16) and a pan-enterovirus (pan-EV) sequence were constructed and used in an external quality assessment (EQA) to determine the performance of laboratories in the detection of EV71 and CA16. The EQA panel, which consisted of 20 samples, including 14 positive samples with different concentrations of EV and either EV71 or CA16 armored RNAs, 2 samples with all 3 armored RNAs, and 4 negative-control samples (NaN(3)-preserved minimal essential medium [MEM] without VLPs), was distributed to 54 laboratories that perform molecular diagnosis of hand, foot, and mouth disease (HFMD) virus infections. A total of 41 data sets from 41 participants were returned; 5 (12.2%) were generated using conventional in-house reverse transcription-PCR (RT-PCR) assays, and 36 (87.8%) were generated using commercial real-time RT-PCR assays. Performance assessments of laboratories differed; 12 (29.3%) showed a need for improvement. Surprisingly, 4 laboratories were unable to detect EV71 RNA in any samples, even those containing the highest concentration of 10(7) IU/ml. Furthermore, the detection sensitivity for EV71 among all laboratories (82.1%) was substantially lower than that for EV (97.4%) or CA16 (95.1%). Overall, the results of the present study indicate that EQA should be performed periodically to help laboratories monitor their ability to detect HFMD viruses and to improve the comparability of results from different laboratories.