Abstract Shiitake (Lentinula edodes Berk) is a white spot mushroom which is found on wood in wild. It is the second most important mushroom in the world. The successful use of non conventional substrate (agricultural residues) for shiitake cultivation is requiring the preparation of a suitable spawn that assures an adequate mycelia growth and development on the substrates. Suitable spawn for this mushroom must be contained mycelia that enable to grow rapidly into the special substrate. For determining the best basal material for production of shiitake spawn, two separate experiments were conducted. In the first experiment different grain seeds like wheat, barley, millet, sorghum in combination with two preparation methods, cooked and soaked in water, were evaluated in factorial design. In the second experiment different sawdust formula that enriched with some other material like wheat bran, rice bran, millet and tea waste were assayed. The experimental design in both experiments was randomized complete block design with 4 replications. Results of the first experiment showed that cooking method is better than soaked method. Among the used grains, the best spawn quality obtained from barley while sorghum had the fastest mycelium growth rate but with low quality. In the second experiment, the best spawn quality was obtained from formula # 1( sawdust, 65% + wheat bran,15% + tea waste 20%) and # 4 (sawdust 80%+ wheat bran,10% + millet 10%). Evaluation of fruit production potential of each spawn need to be determined in the future research.

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