Phytoplasmas are non-cultivable organisms for which biological and molecular characterization is a challenge. Despite the development of new sequencing technologies, few high-quality phytoplasma genomes are available. This research describes the genome sequencing of the phytoplasma involved in grapevine Flavescence dorée, a devastating disease in European vineyards. Analysis of the gene content and comparison with other representative phytoplasmas revealed specific characteristics of the “Flavescence dorée” phytoplasma chromosome. Their implications for the development of the disease are presented and discussed.