The objectives of this study were to understand the sequence variation and the putative protein structure of pipo gene in the Potato virus Y (PVY) collected from Solanum tuberosum. The pipo gene in PVY was cloned using a pair of degenerate primers designed from its conserved region and its sequences were used to re-construct phylogenetic tree in Potyvirus genera by a Bayesian inference method. An expected fragment of 235 bp was amplified in all 20 samples by RT-PCR and the pipo genes in the 20 samples assayed shared more than 92% nucleotide sequence similarity with the published sequences of PVY strains. Among the 20 pipo gene sequences, 13 polymorphic sites were detected, including 4 parsimony informative sites and 9 singleton variable sites. These results indicate that PVY pipo gene is highly conserved but some sequence variations exist. Further analyses suggest that the pipo gene encodes a hydrophilic protein without signal peptide and transmembrane region. The protein has theoretical isoelectric points (pI) ranging from 11.26 to 11.62 and contains three highly conserved regions, especially between aa 10 and 59. The protein is likely located in the mitochondria and has a-helix secondary structure. Bayesian inference of phylogenetic trees reveals that PVY isolates are clustered in the same branch with high posterior probability, while Sunflower chlorotic mottle virus (SoCMoV) and Pepper severe mosaic virus (PepSMV) are closely related, consisting with the classification of Potyvirus genera using other approaches. Our analyses suggest that the pipo gene can be a new marker for phylogenetic analysis of the genera. The results reported in this paper provide useful insights in the genetic variation and the evolution of PVY and can stimulate further research on structure and function of the PIPO protein.