Hand, foot, and mouth disease (HFMD) is a common infectious disease caused by multiple enteroviruses (EVs) in China. To better define the etiologic agents and clinical characteristics of HFMD, we conducted this study in Yunnan, China.In this study, 1280 stool specimens were collected from pediatric patients hospitalized for treatment of HFMD in 2010. EV was detected with nested reverse transcription polymerase chain reaction and directly genotyped by gene sequencing of the viral protein 1 (VP1) region. Phylogenetic analysis was performed based on the VP1 partial gene and the clinical characteristics were analyzed using SPSS Software.Of 1280 specimens, 1115 (87.1%) tested positive for EV. Seventeen different EV serotypes were detected. Coxsackievirus A16 (CA16) was the most frequently detected serotype (615/1115 cases, 55.1%), followed by enterovirus 71 (EV71; 392/1115, 35.2%), CA10 (45/1115, 4.0%), and CA4 (23/1115, 2.1%). Among the 709 severe cases, CA16, EV71, CA10, and CA4 accounted for 48.0%, 42.0%, 3.5%, and 2.3%, respectively. Of the 26 critical cases, 13 were caused by EV71, 9 by CA16, 2 by CA4, and 1 each were the result of CA10 and E9, respectively. All EV71, CA16, CA10, and CA4 isolates were highly homologous to the strains isolated from mainland China, and belonged to the C4a, B1a, G, and C genotypes, respectively.Our study showed that EV71 and CA16 were the main causative agents for severe and critical HFMD, but other serotypes can also cause severe and critical cases.