Abstract: Spawn quality is the most important factor at production edible mushroom. In order to determine the effects of substrates spawn preparation on mycelium growth of oyster mushroom species, the experiment was conducted in a factorial experiment design at randomized completely with three replications. In the experiment, first and second factors respectively were substrates (wheat, corn and millet) and oyster mushroom species (Florida, Citrinopileatus and Ostreatus). The results clearly demonstrated that between various substrates used, maximum and minimum mycelium growth rate were recorded for corn and millet, respectively and between various used species, maximum and minimum mycelium growth rate was at Florida and Ostreatus species, respectively. Also the results showed that spawn dry matter has different considerably after completed maturation and between substrate maximum and minimum dry matter brought by corn and millet substrate, respectively, but it had not significant between various species. Similarly substrates by species interaction showed that the maximum and minimum mycelium growth rate were Florida with corn substrate and Ostreatus with millet substrate, respectively.