Armillaria root and crown rot is a serious and important disease of woody plants. The disease has a universal distribution and causes economical losses on a broad host range of trees in gardens, forests and urban environments. The aerial symptoms of disease are discoloration, yellowing, drying and defoliation of leaves and die back of trees. The signs of Armillaria root and crown rot include formation of white fan mycelium under the bark, black rhizomorphs and occurrence of basidiocarps around the crown of infected trees. The causal agent of the disease is a Basidiomycota fungus, belongs to Tricholomataceae family and the genus Armillaria in which the species Armillaria mellea is well- known among different species for its pathogenicity on trees. Considering the importance of the disease, detection of the pathogen from soils of gardens, nurseries and urban environments, is necessary to establish on IPM program for decreasing and prevention of losses. Numerous methods have been used for the detection of soil-born fungi including semi-selective media, baiting and serological and molecular methods. To achieve a specific, sensitive and rapid detection method for the pathogen in soil and root, the present investigation was conducted. Thirteen isolates belonging to genus Armillaria were obtained via culturing basidiocarps growing around the crown of infected trees and confirmed using universal primers ITS1/ITS4 and specific primers AR1/AR2 in nested PCR. Species specific primers ITS4/AMEL3 were used to identify A. mellea and nine isolates were shown specific band of A. mellea . To study genetic diversity, PCR-RFLP of ITS region amplified with primers ITS4/AMEL3 was carried out, using Hin fI, Alu I and Mse I restriction enzymes. Results showed that only Hin fI could distinguish two groups within isolates of A. mellea . At the next step, the isolates # 295c and A2 were used as inoculum that were previously identified as A. mellea and was inoculated into the soil. For detection of pathogen, soil direct culture method, baiting and nested PCR method were used simultaneously. The culture medium was not capable of detecting pathogen in long-term period. In the baiting method, Pelargonium hortorum rooted cutting was used as bait. In this method, required time to detect pathogen was quite long. In nested PCR method detection of pathogen was done successfully for isolates # 295c within six months and isolate #A2 within three months at different intervals. To verify the accuracy of the used detection method, the nested PCR product was sequenced directly. Results revealed that there is 99% similarity between Isfahan isolate and the isolate A. mellea registered in GenBank. Then the possibility of pathogen detection in wood and soil samples which were collected from gardens and nurseries was tested with soil direct culture method and ne