The hard tick Ixodes ricinus is the principal vector of tick-borne encephalitis virus (TBEV) in Slovenia; but until now, there was no information about the prevalence of TBEV infection in Slovenian ticks. We conducted a 2-year survey in 2005 and 2006, during which we were collecting I. ricinus ticks monthly in eight different locations of Slovenia. A total of 4777 I. ricinus ticks were collected: 1515 in year 2005 and 3262 in year 2006. The collected ticks were pooled into groups from which total RNA was extracted. Viral RNA was detected using real-time RT-polymerase chain reaction (PCR). Ticks infected with TBEV were found in six of eight locations. Viral RNA was detected in 8 of the 230 pools of ticks collected in 2005 and in 14 of the 442 pools collected in 2006. Prevalence of TBEV infection in Slovenian ticks was determined as 0.47%: 0.54% in 2005 and 0.43% in 2006. The detected infection rate in ticks significantly correlates with the TBEV incidence rates in selected areas. Using the method of sequencing, we have confirmed that the TBEV in ticks is genetically related to the TBEV in Slovenian patients.