Abstract: Objective To rapidly detect and identify an outbreak of hand, foot, and mouth disease (HFMD) caused by CV-A6 enterovirus in Heyuan City in May 2013 with molecular biological technology. Methods Specimens of stool and throat swabs of HFMD cases and close contacts were collected and detected for pan-EV, EV-A71, CV-A16, and CV-A6 by real-time PCR. VP1 genetic partial fragments of the positive samples were amplified with semi-nested PCR, and were adopted for sequencing and genetic analysis. Results In this outbreak, 96.15% of cases (25/26) and 28.95% of close contacts (11/38) were pan-EV positive; 96.15% of cases (25/26) and 18.42% of close contacts (7/38) were CV-A6 positive. All samples were negative for EV-A71 and CV-A16 nucleic acid. Partial fragments of VP1 were amplified from 10 samples of 7 cases and 3 close contacts by using semi-nested PCR. Sequence analysis showed that the nucleotide homology of all partial VP1 was 100.0% homologous with CV-A6. Conclusion The outbreak in a kindergarten in Heyuan City of Guangdong Province was caused by enterovirus CV-A6. Combining the real-time PCR and semi-nested PCR can be an effective way for rapid detection and identification of CV-A6 enterovirus.