Objective: The genetic characteristics of the human adenovirus type 53 (HAdV-53) strains isolated from Taiyuan city of Shanxi Province were studied to obtain the baseline data of their molecular characteristics. Methods: Conjunctival swabs (n=79) were collected from epidemic keratoconjunctivitis (EKC) patients in Shanxi eye Hospital in 2016, and five HAdV-53 strains were obtained after virus isolation and identification based on the three major capsid genes sequences including Penton base, Hexon and Fiber gene. And the corresponding sequences of global epidemic HAdV-53 strains and the strains with the same genetic origin as HAdV-53 were also downloaded from GenBank database, and then the three gene database were established, respectively. With the database, phylogenetic tree was constructed, and the genetic and molecular evolutionary characteristics were analyzed with bioinformatics software. Results: Five HAdV-53 strains in Shanxi Province in 2016 showed high consistency with the HAdV-53 strains prevalent in other countries in 1996-2014 (>99.8%). All HAdV-53 strains were in the same evolutionary branch with their recombinant source genotypes (HAdV-37 and HAdV-8) in Penton base and Fiber gene, respectively, and maintained a high degree of consistency in gene sequences. In Hexon gene, HAdV-53 strains were more closed to its recombinant source genotype HAdV-22, the nucleotide and amino acid sequences between two types were highly homologous, while HAdV-53 and HAdV-22 belonged to different evolutionary branches, and the evolution rate of HAdV-53 based on Hexon gene was 3.51×10^-5 substitution/site/year. Conclusion HAdV-53 has become an important new ocular infectious pathogen of Taiyuan. HAdV-53 strain are relatively conservative and stable based on Penton base, Hexon, and Fiber gene.