Armillaria root and crown rot is an important disease of woody plants including fruit, forest and ornamental trees in different regions. The disease is common in Isfahan and causes economical losses on a broad range of fruit trees. There only a few tolerant varieties to disease, and there is no efficient method in controlling the disease. Therefore, detection of the pathogen from soil is necessary to establish an IPM program for decreasing losses. In this study, specific primers were successfully used for rapid, sensitive and specific detection of Armillaria isolates from soil and wood samples. DNA was isolated directly from soil and wood samples and ITS region was amplified using universal primers ITS1 and ITS4 and specific primers AR1 and AR2 in nested PCR. The nested PCR method enables the detection of pathogen directly from soil and root samples without the need for isolation and cultivation of mycelium of Armillaria and in earlier steps of the disease progress.