This study aimed to identify and evaluate potential alternative substrate materials for the cultivation of Lentinula edodes (shiitake mushrooms) to address the growing instability in the supply of oak sawdust. A selection of locally abundant resources, including citrus branches, buckwheat hulls, Cryptomeria japonica sawdust, Miscanthus, and Sasa quelpaertensis, was evaluated as potential substitutes for oak sawdust. The results indicated that Miscanthus is rich in iron and manganese, which are essential cofactors for lignocellulose-degrading enzymes, such as cellulases, laccases, and peroxidases. In vitro mycelial growth assays (column tests) indicated that Miscanthus facilitated the most rapid growth among the alternative substrates for the shiitake strains ‘Chamaram’, ‘L808, ’ and ‘Haleumang’.’ For the strains 'Sanjo 701' and 'Nongjingo’, both oak sawdust and Miscanthus demonstrated comparable mycelial growth rates. Further cultivation tests with the strain ‘L808’ revealed that Miscanthus, whether used independently or in combination with oak sawdust, resulted in cultivation periods, primordia formation, and yield characteristics comparable to those achieved with the oak sawdust medium. These findings suggest that Miscanthus has significant potential as an alternative substrate for shiitake cultivation. However, as the study employed small, laboratory-scale blocks weighing approximately 300 g, while farm-scale blocks typically weigh 1.3–1.5 kg, fruiting was limited to a first flush harvest. Consequently, additional validation using farm-scale blocks is necessary to assess their practical applicability.