HFRS is an endemic disease throughout Croatia. The incidence of HFRS varies in a cyclic fashion, with peaks occurring every couple of years, coinciding with peaks in vole populations. PUUV was shown to be dominant pathogen during the last HFRS outbreak in Croatia in 2002. We focused our research on two newly discovered localities (Okucani and Nova Gradiska) with a high number of reported HFRS cases and a significant increase in rodent population. PUUV infection was verified in 84.2% of patients at this region during the 2002 outbreak. Genetic analysis of wild-type (wt) PUUV strains was performed. Fifty seven bank voles Clethrionomys glareolus originating from PUUV-associated HFRS areas were screened for the presence of PUUV N antigen and 15 (26%) were found positive. Total RNA isolated from rodent lung tissues was reverse transcribed followed by PCR amplification with primers specific for PUUV medium (M) or small (S) genome segments. Partial PUUV M segment sequences (approximately 450 bp long) were recovered from five bank voles and partial S segment sequences (app. 250 nt long)-from two bank voles. Genetic analysis of Croatian wt-PUUV strains revealed their close relatedness suggesting that the two localities belong to the same natural focus of infection. On phylogenetic trees Croatian PUUV strains clustered together with the strains from Slovenia and Austria forming distinct Alpe-Adrian genetic lineage.