Objective The detection results of intestinal virus nucleic acid related to HFMD( hand,foot and mouth disease)from the anal swabs and throat swabs were compared so as to explore more effective methods for laboratory diagnostic of the disease. Methods Real-time fluorescence quantitative polymerase chain reaction (PCR)was used to detect RNA of enterovirus 71(EV71),coxsackievirus A16(CA16),and other enterovirus (EV)in the paired samples. The virus detection results from the two sample types were compared to find out the miss rate in a single sample. Results The detection rates of EV71,CA16 and Non-EV71/Non-CV16 enterovirus (Non-EV71/Non-CV16 EV)in the anus swabs/ throat swabs were 17.25%/12.18%,4.03%/3.54% and 40.28%/45.32%,respectively. Obviously ,the detection rates of EV71 and CA16 in anal swabs were higher than those in throat swabs ,while the detection rates of Non-EV71Non-CV16 EV was lower than that in throat swabs. The differences were statistically significant(P<0.05). Compared with the detection results of the paired samples ,the miss rate of a virus in a single sample type ranged from 10.33%to 36.27%. Conclusion The detection miss rates of HFMD related intestinal virus nucleic acid in one sample type(anal swabs or throat swabs)are high,while tak-ing the detection in the paired samples can significantly improve the detection rate.