Since its first detection in the Netherlands in 1992, Iris yellow spot virus (IYSV, genus Tospovirus) has been reported worldwide in Allium crops, in a few ornamentals and in a small number of weeds. After recent findings of IYSV in Alstroemeria and Eustoma in the Netherlands, a number of neighbouring onion fields were surveyed. In 2005 and 2006, only few infected plants were found with obvious symptoms of IYSV. In 2007, after sampling and testing small leaf samples with various types of damage, including small brown-yellow colored spots and spots with thrips feeding damage, a high percentage of plants were found with positive IYSV scores in ELISA. Infection by IYSV could be confirmed in most ELISA-positive samples by RT-PCR. Under laboratory conditions, evidence was obtained that Thrips tabaci acts as a vector for this virus. Acquisition of the IYSV from infected Datura stramonium plants resulted in virus uptake and replication in over 60% of the thrips exposed, as determined by Western blotting and immunolocalisation of the virus in the foregut, and in epithelial and midgut muscle cells using antibodies against both the viral N and NSs proteins. Successful transmission of IYSV to seedlings of Emilia sonchifolia was observed, however, only at low frequency under the laboratory conditions used.