The genetic structure of Potato virus Y (PVY) populations was investigated in naturally-infected pepper (Capsicum annuum) fields, collected at eight different localities in northern Tunisia, where 23% of the sampled plants were homozygous for the pvr21 recessive resistance allele, while the other plants carried the dominant susceptibility allele pvr2+. Restriction fragment length polymorphism analysis at three PVY genome segments revealed a high level of viral diversity, with a majority of cases showing co-infection of individual plants by several PVY haplotypes and a strong genetic differentiation of viral populations collected in the different localities. Geographic distances affected the differentiation of PVY populations and isolation by distance among these populations was significant. However, the occurrence of the pvr21 resistance allele did not contribute to the structure of viral populations, suggesting that the virulence properties of the virus did not significantly affect its fitness. Consequently, greater deployment of the pvr21 gene would probably not be a suitable strategy to control PVY, and other resistance genes should be preferred