Among various defence strategies used to protect potato plants from a viral infection, a deficiency of the eukaryotic translation initiation factors (eIF) provided by the host-mediated translational suppression has been characterized as a promising approach in modern crop breeding. The strategy used in the present study relies on knocking down the genes of the eIF4E-encoding family in a susceptible potato cultivar by the RNAi silencing mechanism. Taking into account the high concern regarding the potential effects of biotech crops on product safety and public acceptance, no bacterial/viral sequences and selectable marker genes were used. A hairpin construct carrying the fragment of potato eIF4E1 gene was created under control of potato Lhca3 (photosystem I 24 kDa light-harvesting protein) gene promoter and terminator. As a result of marker-free Agrobacterium-mediated transformation, two independent events were identified; one was confirmed to be clean from vector-backbone DNA sequences. Due to high homology between the genes encoding eIF4E1 and eIF4E2 potato factors, the silencing of both genes was achieved. During the two seasonable experiments, the intragenic line with higher production of small RNAs due to the expression of hpRNAi cassette showed a strong level of resistance after the mechanical inoculation with agriculturally significant PVYNTN strain and produced tubers phenotypically similar to non-infected control. Overall, the results obtained here indicate that RNAi-mediated transcriptional regulation of targeted eIF4E gene family using plant tissue-specific promoters in marker-free intragenic potato plants is a promising strategy for improving the viral resistance in clonally propagated crops without affecting plant phenotype and productivity.