D1/D2 domain of 26S rDNA sequence-based alignment and analysis is a widely used method for yeast identification and classification.Rapid and repeatable protocols to extract and prepare the chromosomal DNA of yeast are the basic needs to acquire the templates for following amplification of D1/D2 domain.However,the present methods to extract chromosomal DNA are time-consuming and unable to deal with large-scale samples at the same time.In the present study,a novel rapid and efficient method to identify a large amount of yeast isolates in the same batch was established.The chromosomal DNA required only 20 minutes to prepare and can be directly used as template for PCR amplification.Results of amplified ITS sequences were clear and easy to identify by sequencing analysis.