Apple is noted to be one of the important fruit trees of cold and temperate regions which ranks as the most valuable and economically significant fruit in the world. One of the serious diseases causing heavy loss on apple is known to be caused by Apple chlorotic leaf spot virus ( ACLSV ), which is the most frequent virus infecting apple worldwide. ACLSV is the type member of the genus Trichovirus , family Betaflexiviridae that posses a flexuous, filamentous particle (740–760 nm in length and 12 nm in width).The ACLSV genome consists of single-stranded RNA (ssRNA) (7552 nt) with a poly(A) tract at its 3' end and contains three ORFs that encode a protein containing methyltransferase, papain-like protease, nucleotide triphosphate-binding helicase, and RdRp motifs, a movement protein and a coat protein. ACLSV infected trees, usually show chlorotic leaf spot, leaf rolling and distortion, and fruit deformation. The virus has spread throughout many countries and recently it has been reported from some regions of Iran. The evidence suggests that the virus spreads between regions through the movement of infected propagating material. Therefore routine iections are required to determine the presence and the distribution of the disease in different regions of Iran. Hence, a detection survey was designed to identify ACLSV from infected trees in Isfahan and Western Azarbaijan provinces to provide information on the incidence of the virus in the orchards of these regions and obtain a more comprehensive overview about molecular properties of local isolates of the virus. For this purpose, 114 leaf and floral symptomatic samples were collected from orchards in different areas of Isfahan and western Azarbaijan in 2009-2010. Double stranded RNA was extracted from the samples and analysed by RT_PCR using primers specific for ACLSV (APCLSV-F/APCLSV-R, A52/A53, ACLSV-CPf/ACLSV- CPr). The RT-PCR assays resulted in positive amplification of expected size bands of 677bp, 358 bp and 566 bp respectively, in most of the samples. The virus-specific primer pair (ACLSV-R and ACLSV- CP Apple chlorotic leaf spot virus f) amplified DNA fragment of 792bp from the coat protein gene of ACLSV. The PCR amplicons from the samples, collected from different apple cultivars with diverse symptoms from various regio were subjected to restriction analysis using Taq I and Mse I enzymes. The results provide five different PCR-RFLP patterns among the local strains of ACLSV. The 792bp bp amplified fragment of the coat protein gene from the representative strains of each RFLP pattern were sequenced and compared with other sequences deposited in the GenBank database using the BLAST search software . The alignment obtained at the nucleotide level revealed a high similarity of the local isolates to ACLSV