Control strategies developed for plant viral diseases through breeding programs can be impaired by adaptive response of pathogens. A few years after the deployment in France of improved tobacco genotypes introgressed with alleles of the Potato virus Y (PVY) recessive resistance gene va, necrotic symptoms associated with PVY infection have been reported on these cultivars. Due to the lack of efficient alternative methods to control PVY, the va resistance sources have to be managed according to viral parameters, such as the dynamic of emergence of virulent variants. The effects of va alleles on the evolution of PVY was tested in experimental conditions using a PVY infectious clone and two couples of resistant tobacco near isogenic lines BVA/Bva (0) and FVA/Fva (2) both allelic for the va gene. Infection efficiencies data showed that a single passage on Fva (2) line led to the selection of virulent viral populations able to overcome both va (0) and va (2) alleles. Sequence analyses of va (2) derived PVY variants revealed that the acquisition of the capacity to overcome va (2) resistance is associated with single point mutations at two different nucleotide positions in the central part of the VPg. The described PVY adaptation process to overcome resistance mediated by different va alleles should be considered for the future development of durable and integrated strategies of management of PVY infections in tobacco crops.