To evaluate molecular variability in the 5′ region of the Plum pox virus (PPV) genome, a fragment spanning the C-terminal part of P3, the complete 6K1 and start of the CI gene in 12 Slovak and French PPV isolates was amplified in IC-RT-PCR and sequenced. Computer analysis of obtained and previously published sequences showed rather substantial differences among isolates of PPV-M and D subgroups (percentage of nucleotide identity ranged from 85.65 to 87.68%). There was a relatively low percentage of heterogeneity among isolates within the same subgroup. Amino acid multiple sequence alignment showed a total of 14 conserved subgroup-specific amino acid positions among PPV-M and D isolates in the examined region. The geographical origin of the isolates was not correlated with their phylogenetic clustering. No reasonable correlation could be established between amino acid substitutions and host species and/or biological properties of isolates. The present IC-RT-PCR detection method targeting a 5′ region of the PPV genome, followed by simple RFLP subgroup determination, could enhance conventional CP-based techniques, especially with peculiar isolates. Using this approach, the Slovak isolate BOR-3 was found to be a natural recombinant between a D and an M PPV-type.