Abstract: Objective To analyze the genetic characteristics of Coxsackievirus A4 (CVA4), CVA6, CVA10, CVA16 and Enterovirus A71 (EV-A71) in specimens of hand- foot-mouth disease (HFMD) cases in Dehong Prefecture, Yunnan Province in 2023. To clarify the genotype composition of enterovirus serotypes and CVA4, CVA6, CVA10, CVA16 and EV-A71 prevalent in HFMD, and to provide baseline genotype data for monitoring the dynamic changes of these five virus genotypes.Methods 94 stool specimens of HFMD cases identified as EV-A71, CVA16 and universal positive by real-time fluorescent PCR in Dehong Prefecture in 2023 were first sequenced and typed for VP4/VP2 binding region to determine the virus type. The VP1 gene of each virus was amplified and sequenced in two segments by using the VP1 gene primers of the corresponding groups of enteroviruses (i.e., enterovirus A group, B group and C group) (group A virus primers: 486/488 and 487/489, group B virus primers: 490/492 and 491/493, group C virus primers: 494/496 and 495/497). The VP1 gene reference sequences of CVA4, CVA6, CVA10, CVA16 and EV-A71 virus representative strains were downloaded according to the literature. The VP1 gene phylogenetic tree was constructed by Mega 6.0 software, and the genetic characteristics were analyzed.Results In 2023, a total of 256 hand-foot-mouth stool samples were collected in Dehong Prefecture, of which 94 were positive for nucleic acid by real-time fluorescent RT-PCR, with a positive rate of 36.72% (94/256). Among 94 strains of virus, 83 strains of group A virus had 5 serotypes (17 strains of CVA4,3 strains of CVA6,6 strains of CVA10,49 strains of CVA16,8 strains of EV-A71); there were 9 strains of group B virus and 2 serotypes: 2 strains of Echovirus type 3 (E3), 7 strains of Coxsackievirus B type 5 (CVB5); there were 2 strains of group C virus and 1 serotype: 2 strains of poliovirus 3 (PV3). The genetic characteristics of VP1 region showed that 17 strains of CVA4 were C2 subtype, 3 strains of CVA6 were D3 a subtype, 6 strains of CVA10 were genotype C, 46 strains of CVA16 were B1 a subtype, 3 strains were B1 b subtype, and 8 strains of EVA71 were C4 a subtype.Conclusion In 2023, the pathogenic spectrum of HFMD in Dehong Prefecture was mainly group A virus, of which CVA16 was the highest detection rate virus, accounting for 52.13% (49/94) of the positive samples. Genetic characteristics analysis showed that CVA4, CVA6, CVA10 and EV-A71 were composed of a single genotype (subtype), while CVA16 was composed of two subtypes (B1a, B1b), and B1a was the main epidemic subtype. It is of great significance to continue to strengthen the molecular epidemiological study of HFMD pathogens, especially group A viruses in Dehong Prefecture in the future.