Morels are precious and rare edible mushroom, which contain 61 species. It is difficult to identify the species of morels based on morphological characteristics. In recent years, morels cultivation technology has made great progress, Morchella sextelata has been cultivated in China widely. In this study, a PCR-based approach was developed for identification of Morchella sextelata, based on a 190-bp segment of internal transcribed spacer (ITS). The species-specific primer, MS1F/MS1R, was designed based on the alignment of sequence obtained from the 20 representative cultivated body fruit of Morchella sextelata as well as 10 sequence data available in Genbank. Verifying the species-specific primer pair demonstrated generation of an amplicon from extracted DNA of Morchella sextelata. The primer pair was also applied and tested for five other species of morels and eight common mushrooms from other genus. This novel PCR assay allowed a rapid and effective quality evaluation of Morchella sextelata.