Abstract: Although it has been widely used in the polyploid breeding of fruit trees, ploidy identification by flow cytometry facilitated in pear trees has not been well reported. In this study, key links in flow cytometry experiment were systemically optimized using pear leaves as materials. The results showed that the young leaves from the third-to-fifth leaf of the spring shoots, especially grew in greenhouse, were better for flow cytometry test. Before xperiment, successively washing young leaves with water and distilled water, and then immersing in distilled water for 10 min was also benefit for the test. In addition, the processes using WPB buffer to extract cell nucleus, twice filtrating the solution with 500 mesh filter membrane and once centrifugation of cell lysis solution were optimal for the ploidy identification by flow cytometry. In total, a simple, rapid and efficient method of flow cytometry suitable for pear ploidy identification was established and could be generally used for pear polyploid identification.