We evaluated the temporal signal and substitution rate of tick-borne encephalitis virus (TBEV) using 276 complete open reading frame (ORF) sequences with known collection dates. According to a permutation test, the TBEV Siberian subtype (TBEV-S) data set has no temporal structure and cannot be applied for substitution rate estimation without other TBEV subtypes. The substitution rate obtained suggests that the common clade of TBEV (TBEV-common), including all TBEV subtypes and louping-ill virus (LIV), is characterized by the lowest rate (1.87 × 10−5 substitutions per site per year (s/s/y) or 1 nucleotide substitution per ORF per 4.9 years; 95% highest posterior density (HPD) interval, 1.3–2.4 × 10−5 s/s/y) among all tick-borne flaviviruses previously assessed. Within TBEV-common, the TBEV European subtype (TBEV-E) has the lowest substitution rate (1.3 × 10−5 s/s/y or 1 nucleotide substitution per ORF per 7.5 years; 95% HPD, 1.0–1.8 × 10−5 s/s/y) as compared with TBEV Far-Eastern subtype (3.0 × 10−5 s/s/y or 1 nucleotide substitution per ORF per 3.2 years; 95% HPD, 1.6–4.5 × 10−5 s/s/y). TBEV-common representing the species tick-borne encephalitis virus diverged 9623 years ago (95% HPD interval, 6373–13,208 years). The TBEV Baikalian subtype is the youngest one (489 years; 95% HPD, 291–697 years) which differs significantly by age from TBEV-E (848 years; 95% HPD, 596–1112 years), LIV (2424 years; 95% HPD, 1572–3400 years), TBEV-FE (1936 years, 95% HPD, 1344–2598 years), and the joint clade of TBEV-S (2505 years, 95% HPD, 1700–3421 years) comprising Vasilchenko, Zausaev, and Baltic lineages.