The complete genomes of three Czech isolates VIRUBRA 1/045, VIRUBRA 1/046, and VIRUBRA 1/047 of Potato leafroll virus (PLRV) were sequenced and compared with 13 complete sequences of PLRV isolates available in GenBank. Among the Czech isolates, VIRUBRA 1/046 and 1/047 showed the highest nucleotide (nt) identity (98.7%). PLRV was the most conserved virus in both open reading frames (ORFs) 3 and 4. The most variable regions were ORFs 0 and Rap1. Interestingly, isolate VIRUBRA 1/045 significantly differed from the other two Czech isolates in ORFs 0 and 1. Moreover, we identified mutations in the amino acid (aa) sequences, which were specific for the Czech isolates. Phylogenetic analysis based on ORF0 showed that the Czech isolates could be classified in two of the three groupings of the phylogenetic tree obtained. This is the first report on sequence analysis of the genome sequences of PLRV isolates from the Czech Republic.