Tembusu virus (TMUV) is a contagious pathogen from fowl that mainly infects ducks and geese, causing symptoms of high fever, loss of appetite, retarded growth, neurological symptoms, severe duck-drop syndrome, and even death. During an epidemiological investigation of TMUV in Northern China, we isolated 11 TMUV strains from ducks, chickens, geese, sparrows, and mosquitoes (2011⁻2017). Phylogenetic analysis of the open-reading frames of genes revealed that these strains clustered into Chinese strains II. The nucleotide and amino acid homologies of NS1 of the strains ranged between 85.8⁻99.8% and 92.5⁻99.68%, respectively, which were lower than those of E (86.7⁻99.9% and 96.5⁻99.9%, respectively), NS3 (87.6⁻99.9% and 98.2⁻99.8%, respectively), and NS5 (86.5⁻99.9% and 97.8⁻99.9%, respectively). Predictions of the tertiary structure of the viral proteins indicated that NS1 in 4 of 11 strains had a protein structure mutation at 180TAV182 that changed a random crimp into an alpha helix. The protein of 6 of 11 strains had a glycosylation site mutation from NTTD to NITD. Furthermore, epidemiological data suggested that TMUV has been circulating in half of China's provinces (17 of 34). Our findings, for the first time, have identified the NS1 protein as a potential hypervariable region for genetic evolution. Additionally, the territorial scope of the virus has expanded, requiring strict bio-security measures or a multivalent vaccine to control its spread.