In order to investigate the Iris yellow spot virus (IYSV) Sampling from onion fields and greenhouse of Khorasan Razavi province in summer of 2009 was done. Totally 435 samples were collected from this onion fields and 142 from ornamental plants (Rose, Gladiol, Iris, Plargonium, Chrysanthemum, Begonia, Petonia and Carnation). The plants that had symptoms such as chloratic, necrotic and diamond shape lesions were collected and transferred under cold condition to labratory then tested by DAS-ELISA and the sap of posetive plants inoculated to 4 coultivar of indicator plants that cultivated in greenhouse Nicotiana rustica (leef deforming and systhemic chloratic and necrosis),N. benthamiana, N. clevelandii, N.tabacum var Samson (systhemic chloratic and necrosis) then the inoculated indicator plants tested by DAS-ELISA, sap of positive indicator plants inoculated to 4 cultivars of onion include in yellow of Neishabour, white of Neishabour, red of Dargaz, red of dorche of Isfahan. The symptoms similar to the symptoms on infected onion in the fields appeared in their plants. For molecular detection, RNA extraction was done by PEG6000 Precipitation and RNX ™)plus ( kit.In RT-PCR tests, specific primers designed for nocleoprotein gene amplified 181 bp and 139 bp fragments.. DAS-ELISA test results have indicated that all of the onion fields were infected with the virus in various rates. IYSV has been detected in 107 samples of onion, 7 samples of chrysanthemum floweres and 1 sample of iris floweres. This is the first repot of Iris yellow spot virus on onion and chrysanthemum in Iran.