AbstractTick‐borne encephalitis virus (TBEV) is a severe problem in Estonia. In the present article the first genetic analysis of Estonian TBEV strains is described. In total, seven TBEV strains were isolated from ticks (Ixodes ricinus and I. persulcaus), rodents (Apodemus agrarius and Cletrionomys glareolus), and serum from a tick‐borne encephalitis (TBE) patient. The nucleic acid sequences of the viral genome encoding almost the complete E protein (nt 41–1250) and the 3′‐NCR‐termini of the Estonian TBEV strains were determined by direct sequencing of RT‐PCR products. The results showed that all three known TBEV subtypes, Western TBEV (W‐TBEV), Far‐Eastern TBEV (FE‐TBEV), and Siberian TBEV (S‐TBEV), co‐circulate in Estonia. The Estonian TBEV strains of the S‐TBEV and W‐TBEV subtypes clustered with the previously reported strains from Latvia and Lithuania. Within the FE‐TBEV subtype, however, the Estonian strain Est2546 clustered together with the strain Sofjin, originating from the Far‐East of Russia, but not with the strain RK1424, isolated in the neighboring Latvia. This suggests a different evolutionary history for the Estonian and the Latvian strains in the FE‐TBEV subtype. The Estonian TBEV strain (Est3535), which belonged to the S‐TBEV subtype, had an organization of the 3′‐NCR similar to that of strains from the Far‐East of Russia (Irkutsk). The 3′‐NCRs of Estonian strains of the W‐TBEV subtype (Est3051, Est3053, Est3476, and Est3509) were very similar to those of the strain Ljubljana I from the Balkans. In the 3′‐NCR sequence of the Estonian strain Est2546, which belonged to the FE‐TBEV subtype, a deletion from position 10461 to 10810 extending approximately 10 nucleotides into the core element, was detected. J. Med. Virol. 74:580–588, 2004. © 2004 Wiley‐Liss, Inc.