Tick-borne encephalitis virus (TBEV) is a significant tick-borne flavivirus responsible for severe human diseases. Here, we analyzed the genetic diversity and evolutionary dynamics of TBEV using 263 genome sequences from the NCBI database and identified key amino acid mutations. TBEV sequences were classified into five genotypes-Baikalian, European, Far-Eastern, Himalaya, and Siberian-showing ORF nucleotide similarity of 81.5% to 88.0% and amino acid similarity of 93.0% to 96.4%. Extensive recombination between genotypes was not observed. Entropy analyses revealed highly variable sites distributed across the Baikalian (n = 2), European (n = 3), Far-Eastern (n = 5), and Siberian (n = 13) genotypes. Each genotype exhibited specific amino acid mutations. Positive selection analysis identified sites under selection in the full dataset (n = 2), as well as in the European (n = 6), Far-Eastern (n = 7), and Siberian (n = 4) genotypes. By integrating highly variable sites, shared genotype-specific mutations, and positively selected sites, we identified 37 key amino acid positions, primarily located on the surfaces of viral proteins. These positions may have a potential impact on protein function and pathogenicity, though further studies are required to validate and evaluate these effects comprehensively. This study provides the first comprehensive analysis of mutational landscapes across TBEV genotypes, uncovering potential critical mutations that may shape viral biology and pathogenicity, and offers valuable insights for further exploration of TBEV characteristics.