Abstract: Objective To study the molecular epidemiology of enterovirus associated with children hand-foot-mouth disease (HFMD) in Kunming from 2015 to 2016 and to explore the correlation between the genetic variation and spatial-temporal distribution pattern of coxsackievirus (CVA16) VP1 gene. Methods Throat swabs and rectal swabs were collected from children included in this study. Reverse transcription polymerase chain reaction was conducted to amplify the full-length VP1 gene of enterovirus 71 and CVA16. We randomly selected 9 CVA16 positive amplicons in 2015 for sequencing and these sequences were used to construct phylogenetic trees with relevant reference sequences from database. Correlation and regression analyses between genetic distances and the isolating times of CVA16 strains were performed. Mantel test between genetic distances and the geographical distances among geographical populations was also carried out. Results Most of the children were infected by CVA16 (the detection rate of throat swabs and rectal swabs were 29.41% and 15.69% respectively). The ratio of CVA 16 infection rate to enterovirus 71 infection rate was 2.5:1 and 2.0:1. Phylogenetic analyses of the VP1 gene proved that all the 9 CVA16 Kunming isolates belonged to genotype B, subgenotype B1b, and it can be further divided into two sub clusters. Phylogenetic and statistical analyses revealed a significant positive correlation between the genetic variation of CVA16 virus and both of the passage of time and geographical distance(all P<0.05). Conclusions The above results demonstrated that the 9 CVA16 isolates from Kunming, Yunnan Province, China in 2015, belonged to new mutant strains, suggesting its unique epidemic characteristic in Kunming city.