Abstract: Objective To provide an overview of the prevalence and etiologic distribution of hand, foot and mouth disease (HFMD) in Shiyan, Hubei, and optimize the detection program, providing the basis for understanding of the epidemiological characteristics and development of control and prevention strategies of HFMD. Methods Clinical data of diagnosed HFMD cases from 2008 to 2009 were analyzed. Herpes samples and throat swabs were collected from 233 patients in the city for RNA extraction. RT-PCR or fluorescent quantitative real-time PCR were carried out for nucleic acid identification and typing. Results HFMD was prevalent in the city throughout the year. CA16 and EV71 were the predominant pathogens. The epidemic was mostly present from May to July, affecting mainly scattered children or kids in kindergartens aged less than 5 years. Higher detection rate was found in herpes samples than in swabs, and by fluorescent quantitative PCR than by RT-PCR. Conclusion Varying degrees of HFMD prevalence were present in multiple counties and districts of the city, with CA16 and EV71 the predominant pathogens. Prevention and control of HFMD should be strengthened for kindergartens in the city from May to July. It is highly recommended to collect suspects herpes samples, or throat swabs as an alternative, for nucleic acid detection using fluorescent quantitative PCR.