Flavescence dorée (FD) is the most threatening grapevine yellows (GY) disease in Europe. Despite strict control measures, alarming signs of the spread of the disease in viticultural areas continue to be detected. FD is attributed to infection by phytoplasma strains of an incidentally cited species, ‘Candidatus Phytoplasma vitis’. In 2017, a GY field survey was carried out in traditional viticulture areas of Tuscany, central Italy. FD phytoplasma (FDp) was detected in 85 GY symptomatic vines, accounting for 17% of a total of 500 symptomatic samples screened. The FDp-positive vines were scattered in 50 vineyards across seven Tuscan provinces, indicating the distribution of FDp has further extended to central and southwestern parts of Tuscany including Florence and Livorno. Multilocus sequence typing of 15 representative FDp strains from six affected vineyards revealed that the Tuscan FDp strains constitute a highly homogeneous lineage within the subgroup 16SrV–C (FD-C). Single nucleotide polymorphisms (SNPs) were identified in the 16S rRNA, rp, and secY genes of the Tuscan FDp lineage. Such SNP markers provide clues to understanding the genetic relationships among different FDp lineages present in Europe and are useful for searching potential vectors and reservoirs involved in the spread of the FDp in the Tuscan region. •Flavescence dorée (FD) is a destructive grapevine disease in Europe.•FD disease has been detected in Florence and Livorno, Italy for the first time.•The FD phytoplasma strains found in Tuscan vineyards are highly homogenous.•Single nucleotide polymorphism markers of the phytoplasma lineage are identified.