Abstract: In order to test whether pyrosequencing of short sequences can be applied to identify Potato virus Y (PVY) strains, thirty whole PVY genome sequences representing nine PVY strains were retrieved from the NCBI database. Sequences of their P1 and CP proteins were compared and a distinctly different region was found out from the sequence of each protein. Both regions were used to design primers for reverse transcription, PCR and pyrosequencing. Two potato leaf samples were collected from Keshan and Nehe of Heilongjiang Province of China. Total RNA was extracted. Reverse transcription and PCR were carried out. The PCR products were applied as templates for pyrosequencing. The pyrosequencing results showed that both leaf samples were infected by PVYN-Wi. This research confirms that the primers we designed can be utilized to identify PVY strains in infected potato plants by pyrosequencing of short sequences.