In this work, the infection of Grapevine fanleaf virus (GFLV) disease was described on the base of symptomatic differences within eight grapevine plants of six grape ciltivars with positive tests on GFLV Among them, cultivars Kodrjanka, Pamjati Negrula, KiSmiS LuSistyj were planted in wine region of the South Moravia (Czech Republic), three infected grapevine cultivars (URS, Cinsaut, Dimrit) included in this study originating from Italy. Except symptomatic evaluation, the differences between isolates were emphasized at the genetic level too, exactly in the frame of RNA2 genomic region coding movement protein. The variability of the tested isolates within the eight plants was in the range from 86.59 to 97.61% at the nucleotide level. The results confirmed very high degree of similarity between virus isolates of GFLV within studied RNA2 region. This fact was assessed by the phylogenetic analysis of obtained sequencing data too.