An extensive study was conducted during 2007 and 2008 in three major tomato production areas of Cyprus, where Tomato yellow leaf curl virus (TYLCV) is commonly found, to assess the incidence and prevalence of naturally infected weed species that could serve as TYLCV reservoirs. Approximately 4,000 of the most common dicotyledonous plants belonging to 122 species from 25 families were collected, identified, and tested for TYLCV presence using serological and molecular methods. The tests included a previously reported conventional polymerase chain reaction (PCR) assay and a real-time TaqMan PCR assay developed and optimized in this study. Real-time PCR was found to be the most sensitive technique, and enabled the detection of TYLCV in 461 samples of 49 different species belonging to the families Amaranthaceae, Chenopodiaceae, Compositae, Convolvulaceae, Cruciferae, Euphorbiaceae, Geraniaceae, Leguminosae, Malvaceae, Orobanchaceae, Plantaginaceae, Primulaceae, Solanaceae, Umbelliferae, and Urticaceae. The results further indicated that the host range of TYLCV in Cyprus is far more extensive than previously documented and, therefore, new management strategies are required. These should focus on the control of alternative virus hosts during the growing season and in crop-free periods.