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Apple chlorotic leaf spot virus (ACLSV) is one of the most common viruses infecting apple and pear trees, and Iran is among the top ten apple-producing countries in the world. In the present study, incidence and genetic diversity of ACLSV were investigated in the main apple-growing regions of Iran. To achieve this purpose, a total of 1053 leaf samples were collected from orchards located in nine Iranian provinces. ACLSV infection was detected by DAS-ELISA in 48 samples (4.55%) from seven provinces and was confirmed by RT-PCR. Based on the geographical origin, 19 representative isolates were selected for phylogenetic analysis. Nineteen amplicons, with a size of 677 base pairs (bp), containing the 3′ end of the movement protein (MP), the coat protein (CP) gene, and 3′-UTR sequence, were sequenced. Sequence analysis, using data of 45 isolates from 12 different countries, including the 19 Iranian isolates, showed that CP gene among the Iranian isolates were 80–99% identical at both nucleotide and amino acid levels, and these isolates were placed in the B6 (AB326224) and P-205 (D14996) ACLSV groups. This is the first genetic analysis of ACLSV in Iran. [ABSTRACT FROM AUTHOR]