Objective To analyze the genetic evolution and hemagglutinin (HA) gene mutation sites of influenza B virus in Chengdu. Methods Influenza virus was isolated from patient's throat swab samples using Madin-Darby canine kidney (MDCK) cells in vitro. The HA gene of influenza B virus was obtained by PCR and was sequenced. The phylogenetic tree was constructed and mutation sites were analyzed by online comparison with NCBI database and MEGA 6.06 software. Results One strain of influenza B virus was isolated by MDCK cells, and 1 755 bp full-length HA gene was obtained by PCR amplification using the nucleic acids of the throat swab and the isolated strain as templates. The obtained sequence was submitted to the GenBank database, and the gene accession number was MH236281. By online alignment and phylogenetic tree construction, the virus infected in this case was confirmed to be Yamagata type B influenza virus. There were 57 HA point mutation bases as compared with Influenza B/Yamagata/16/88 (GenBank No. M36105). There were 20 mutated bases in HA as compared with the World Health Organization (WHO)-recommended vaccine strain Influenza B/Utah/08/2014 (GenBank No. KU592766). HA1 amino acid mutation site was further analyzed. Compared with the epidemic strains in Sichuan in recent years, HA1 amino acid sites have undergone varying degrees of mutations, of which there were only 4 point mutations compared with Wenjiang/2010 isolate (GenBank No. KP461138). Compared with the WHO-recommended vaccine strain Influenza B/Utah/08/2014, two amino acid sites mutated (L176Q and M255V), but the mutation was not located in the antigenic determinant region of HA1. Among them, site 176 was a new mutation. The amino acid was leucine (L) at site 176 of HA1 epidemic strains in Sichuan, Influenza B/Yamagata/16/88, and the WHO-recommended vaccine strain Influenza B/Utah/08/2014, whereas the amino acid was glutamine (Q) in the influenza B strain isolated in this study. Conclusion Mutations have occurred in the HA gene of influenza B virus infected in Chengdu during 2017-2018, while these mutations have not yet caused antigenic changes.