The diversity of European stone fruit yellows (ESFY) phytoplasmas was studied using a multilocus typing approach to verify possible variability related to phytoplasma population modification/diversification. After their identification at the 16S ribosomal level about 70 ESFY strains were selected for further characterization using amplicons from ace, imp and secY genes as molecular markers. Field collected samples from diverse stone fruits, reference strains in periwinkle before micropropagation or micropropagated since up to 15 years, and a strain from pineapple were employed. Characterization was performed by RFLP analyses and sequencing of selected amplicons obtained in nested PCR assays. A clear differentiation of the strain infecting pineapple in Colombia from all others was observed by both, differential restriction profiles on all the three genes and SNPs presence in the ace gene sequence alignment. The RFLP analyses of strains from field collected material showed that the profiles of strains before micropropagation correspond to four out of the 18 different lineages detected in the field-collected material. Moreover also the strains maintained in micropropagation were differentiable from the corresponding strains before micropropagation, and resulted differently represented in the field-collected samples. In particular only one lineage was consistently retrieved in all the different materials, while one lineage was present only in micropropagated strains and another in micropropagated and field collected samples. This is a clear indication of rearrangement in those genes in the diverse samples according to differential plant host (pineapple, stone fruits, periwinkle), growing system and time frame. In particular the pineapple strain showed differential RFLP profiles on ace and secY genes, in the first case confirmed also by the presence of SNPs in the sequence, indicating possible adaptation of this strain to the new host plant in which ESFY phytoplasma was detected for the first time.