In this study, we investigated the amino acids content of shiitake mushrooms grown in eight different media and under three different temperature conditions. The sawdust media were supplemented with 4 kinds of nutrients - rice bran, barley, bean curd refuse, and red ginseng refuse in an 8:2 ratio with or without 0.5% calcium chloride, and the fruiting temperature was set to low (13–15oC), medium (18–20oC), or high temperatures (24–26oC). Seventeen amino acids were detected, and the content varied depending on the compositions of the media. The highest total amino acid content (209.7 mg/g) was measured in mushrooms from the media with red ginseng refuse, and the glutamic acid content was the highest (51.3 mg/g) in mushrooms from the media with barley. In terms of fruiting temperature, the amino acid content was the highest (38.2%) at medium temperature followed by the content at low (32.5%) and high temperature (29.3%). The amino acid composition of shiitake mushrooms was similar between the medium and low temperature conditions.