The objective of this study is to establish an accurate and high efficiency method for Ping’ou hybrid hazelnut cultivar identification. 43 cultivars/varieties of Ping’ou hybrid hazelnut that were introduced throughout China were used in this experiment. 12 polymorphic EST-SSR markers that developed in the previous study were used to select the high efficient marker combination for cultivar identification. The result showed that: the PIC value of the 12 EST-SSR markers among the 43 samples ranged from 0.3800 to 0.7839, with the average value of 0.5113. All the markers showed good polymorphism and amplification result when visualized by capillary electrophoresis. The genetic relationship of Ping’ou hybrid hazelnut showed very complex due to their breeding method. The genetic distance of Bokehong and Ping’ou 62, Ping’ou 3 and Ping’ou 7, Ping’ou 30 and Ping’ou 48 were the lowest among all the samples. Finally, the combination of four EST-SSR markers, CAF-2, CAF-3, CAF-12 and CAF-13, could identify 43 cultivars/varieties of Ping’ou hybrid hazelnut, the combination of CAF-2 and CAF-13 could identify 16 main cultivars/varieties. The result offered an accurate, convenient and efficiency method for cultivar identification of Ping’ou hybrid hazelnut and could also offer reference for the relative researches.