The tick-borne encephalitis virus (TBEV) has expanded its distributional range in Europe over recent decades. Italy is considered a low tick-borne encephalitis (TBE) incidence country, nonetheless, human cases have recently increased, reaching their maximum so far in 2022. This study reports on a new TBE hotspot in the Autonomous Province of Trento (Italy), along with a description of TBEV genetic variants at the provincial level. Tick sampling was performed where several TBE human cases were reported in 2022. As a result, 458 Ixodes ricinus ticks were collected (38 females, 49 males, 371 nymphs) and screened for TBEV through real-time PCR, pooling the nymphs while analyzing the adults individually. Eight samples were found to be positive for TBEV, corresponding to a 4.6 % prevalence in adults and a 1.1 % minimum infection rate in nymphs in the area. The identified TBEV genetic variants were compared with other TBEV European subtype (TBEV-Eu) strains. The relatively high prevalence in ticks and the reported human cases beyond the year of vector collection suggest a sustained virus circulation. Additionally, phylogenetic analysis of the genetic variants in the Trentino Province shows a separation among eastern and western sites. This study highlights the crucial importance of active surveillance for mitigating TBE risk, especially in highly anthropized Alpine areas.