The seasonal variations of grapevine yellow phytoplasma were investigated in four phloem-feeding planthopper and leafhopper species that are vectors of plant disease agents. In total, 1,148 wild specimens were collected from three vineyard agroecosystems in the Marche region (central-eastern Italy), from May to September 2008, and analyzed using polymerase chain reactionrestriction fragment-length polymorphism methods. Of 525 Euscelis lineolatus Brullé, 25.1% were positive for aster yellow phytoplasma (16SrI-C, 16SrI-B subgroups) and stolbur phytoplasma (16SrXII-A subgroup; Vergilbungskrankheit type I [VK-I]). Of 368 Hyalesthes obsoletus Signoret, 19.3% were positive for the 16SrXII-A subgroup (VK-I, VK-II; mainly according to their host plant). Of 146 Neoaliturus fenestratus (Herrich-Schäffer), 15.1% were positive for the 16SrI-C and 16SrI-B subgroups, and 7.3% of 109 Psammotettix alienus (Dahlbom) were positive for the 16SrI-B subgroup. The total inoculation efficiency in the feeding medium assays was 57.1% for P. alienus, 44.7% for E. lineolatus, 44.4% for N. fenestratus and 33.9% for H. obsoletus. All of the phytoplasma subgroups identified in the insect bodies were also detected in their feeding media. Detection of stolbur phytoplasma in E. lineolatus feeding media strengthens the hypothesis that it is a candidate vector of Bois noir disease causal agent. The phytoplasma subgroups detected in the Auchenorrhyncha species showed variations according to season and/or vineyard agroecosystem. This study highlights the different specificities of these phytoplasma-Auchenorrhyncha species relationships, and suggests a primary role of the entire vineyard agroecosystem in the epidemiology of grapevine yellow phytoplasma diseases.