Shiitake mushrooms (Lentinula edodes) hold significant cultural and economic value, particularly in Asia where they are extensively cultivated. The diversification of shiitake cultivars, driven by the need to adapt to various climatic conditions and cultivation methods, has resulted in over 200 distinct cultivars. Reliable identification of these cultivars is crucial for breeding, intellectual property protection, and effective genetic resource management. Traditional morphological methods are inadequate due to their subjectivity and labor-intensive nature. This study leverages nanopore high-throughput sequencing to comprehensively analyze the rDNA regions (SSU, ITS, LSU, IGS) of 41 shiitake strains from Taiwan’s Bioresource Collection and Research Center (BCRC), comprising 5 wild strains, 33 commercial strains, and 3 wild-commercial hybrids. Our results identified the IGS1 region as the most variable and suitable for cultivar differentiation. Consequently, we developed an interactive online database that integrates 317 IGS1 sequences from Taiwan, Japan, and China. This platform allows users to upload their IGS1 sequences and identify similar cultivars through a user-friendly interface, enhancing the precision and efficiency of shiitake cultivar identification.