Small interfering RNAs (siRNAs) produced from template double-stranded RNAs (dsRNAs) can activate the immune system in transgenic plants by detecting virus transcripts to degrade. In the present study, an RNA interference (RNAi) gene silencing mechanism was used for the development of transgenic potato plants resistant to potato virus Y (PVY), the most harmful viral disease. Three RNAi gene constructs were designed based on the coat protein (CP) and the untranslated region parts of the PVY genome, being highly conserved among all strains of the PVY viruses. Transgenic potato plants were generated using Agrobacterium containing pCAMRNAiCP, pCAMRNAiUR, and pCAMRNAiCP-UR constructs. The transgene insertions were confirmed by molecular analysis containing polymerase chain reaction (PCR) and southern blotting. The resistance of transgenic plants to PVY virus was determined using bioassay and evaluating the amount of viral RNA in plants by RT-PCR, dot blotting of PVY coating protein, and enzyme-linked immunosorbent assay (ELISA). Bioassay analysis revealed that more than 67% of transgenic potato plants were resistant to PVY compared with the non-transgenic plants, which showed viral disease symptoms. No phenotypic abnormalities were observed in transgenic plants. Out of six lines in southern blot analysis, four lines had one copy of the transgene and two lines had two copies of the target genes. No correlation was detected between the copy number of the genes and the resistance level of the plant to PVY. Transgenic lines obtained from all three constructs indicated more or less similar levels of resistance against viral infection; however, CP-UR lines exhibited relatively high resistance followed by CP and UR expressing lines, respectively. Meanwhile, some lines showed a delay in symptoms 35 days after infection which were classified as susceptible.