Objective:To perform adenovirus detection and genetic evolutionary analysis on specimens from a fever outbreak in Kunming city.Methods:Pharyngeal swabs from typical febrile patients were collected and tested for nucleic acids of 30 common respiratory pathogens using TaqMan Array Card technology. The full-length sequences of three important genes of adenovirus, Penton base, Hexon and Fiber, were amplified, sequenced and typed using Nanopore high-throughput sequencing. A phylogenetic tree was constructed. Molecular variations and genetic evolution of the three genes were analyzed.Results:Five specimens were collected and all of them tested positive for adenovirus and Haemophilus influenzae. The sequences of the full-length coding regions of the Penton base, Hexon and Fiber genes were obtained by Nanopore sequencing. The homology of the three gene sequences in the five specimens was 100.0%, 99.9%-100.0% and 100.0% in nucleotide sequences, and 100.0% in amino acid sequences. The three genes in the specimens had the highest homology with those of the reference strain of human adenovirus type 3 (HAdV3, accession number: AY599834) in nucleotide sequences, which was 98.6%, 98.7% and 98.9%, respectively. Results of the phylogenetic analysis of the three genes were basically consistent. These Kunming strains were clustered into an independent clade with the reference HAdV3 strain and had a distant relationship with the strains isolated in foreign countries and Taiwan, China in the early years. They were closely related to the domestic and foreign strains in recent years and highly homologous to the 2019 Japanese strain (accession: LC703523) and the Guangzhou strain (accession: MZ540961). Compared with the reference strain, these Kunming strains had five amino acid variations in Penton base, 10 in Hexon and 11 in Fiber. Conclusions:All of the adenovirus strains isolated in this outbreak belong to P3H3F3 type based on the full-length sequences of Penton base, Hexon and Fiber genes. They share high homology with the domestic and foreign HAdV3 strains, including the reference strain. Compared with the reference strain, several amino acid mutations are identified in these Kunming strains, and most of them are in the high variability region or functional regions. M7L in the Hexon protein is an unique amino acid mutation site of Kunming strains.