‘Candidatus Phytoplasma (Ca. P.) solani’ is associated with Bois noir (BN) of grapevine and stolbur of solanaceous plants and is primarily transmitted by Hyalesthes obsoletus Signoret. Four tuf-a and five tuf-b1 ‘Ca. P. solani’ strains were transmitted to tomato plants (cv. Micro-Tom) to set the basis for studying molecular interactions between different strains of the pathogen and host plants. The strains were acquired by using bait-plants and by capturing H. obsoletus adults on bindweed and stinging nettle in vineyards of Friuli Venezia Giulia (northeastern Italy) with a high prevalence of BN. Captured insects were forced to feed on healthy tomato plants to induce infection. All strains obtained from symptomatic plants and confirmed by real-time PCR were maintained on tomato through grafting. Successively, the strains were characterised by macroscopic and microscopic symptoms induced in the host, Multi-Locus Sequence Typing (MLST) based on tuf, secY, stamp, and vmp1 genes, in-planta spread and multiplication patterns. Molecular typing distinguished the strains into five lineages comprised in three clusters: one including strains of tuf-a genotype and two including strains of tuf-b1 genotype. Quite different symptoms were induced on tomatoes by strains belonging to the two tuf genotypes; infection by tuf-a strains resulted in plant decline around 95–100 days after grafting and absence of cauliflower-like inflorescence with symptoms of phyllody and virescence, which were usually associated with tuf-b1 strains. The different symptoms, the outcome of disease, and the ultrastructural observation performed on sieve elements suggested a higher virulence of tuf-a strains in tomato. Overall, our results propose that genomic variability of ‘Ca. P. solani’ strains should be extensively explored to determine possible associations with type of symptoms and strain virulence.