Apple Chlorotic Leaf Spot virus (ACLSV) was isolated from symptomless peach trees and then identified with a specific antiserum (Sanofi comp) using Double Antibody Sandwich ELISA (DAS-ELISA) and Dot Immunobinding Assay (DIBA). Survey was carried out during 2001 to 2003 in different locations on commercial peach orchards. Percentages of infection were 6.3, 6.7, 12.3 and 10.9 in Menofia (Khatatba) . EL-Behira (EL-Nobaria North Sinai (Rafah) and Dakahlia (Metghamr), respectively. ACLSV was transmitted mechanically from peach trees those showing less vigorous and some abnormalities on growth without leaf symptoms to different herbaceous hosts including Chenopodium quinoa wild, Ch. amaranticolor (Cost&Reyyn) and Phaseolus vulgaris L cv. Pinto. Typical chlorotic spots were appeared on the woody indicator GF305 leaves one month after chip-budding or infected buds. Reverse transcription polymerase chain reaction (RT-PCR) procedure was used to amplify fragments using coat protein gene primers (358bp) of the viral coat protein gene. PCR product was used to generate ACLSV-specific probe to detect the virus in infected plants using non-radioactive molecular hybridization methods. The result showed that it is more sensitive than DASELISA and can be used for large scale detection. PCR product was cloned and sequenced. Comparison between local isolate sequence and other published sequences of the Same virus shows 79.4% - 84% homology.