Hand, foot and mouth disease is a common illness in children and is usually caused by coxsackievirus A 16 and enterovirus 71. It has been noted that enterovirus 71 infection is more severe with significantly greater frequency of serious complications and fatality than coxsackievirus A 16. Therefore, it is important to develop a rapid and specific assay for discriminating coxsackievirus A 16 and enterovirus 71 in hand, foot and mouth disease outbreaks. In this study we designed two sets of RT-PCR primers specific for coxsackievirus A 16 and enterovirus 71. One hundred and eighty-nine viruses were evaluated for this molecular diagnosis assay. Among 110 enterovirus 71 strains, the enterovirus 71 specific primers gave clear signal for 107 clinical enterovirus 71 isolates and three reference enterovirus 71 strains. None of coxsackievirus A 16, other enteroviruses or non-enteroviruses show signal for enterovirus 71-specific primers. On the other hand, among 28 coxsackievirus A 16 strains, the coxsackievirus A 16-specific primers detect 27 clinical isolates and one reference strain but show no cross-reaction with other viruses. The molecular assay developed in this study provides a sensitive and specific way to distinguish coxsackievirus A 16 and enterovirus 71 induced hand, foot and mouth disease, which will be a useful rapid diagnostic method in future outbreaks.