In order to improve typing methods for phytoplasma belonging to the 16SrX phylogenetic group, isolation of new genetic markers was needed. A double suppression subtractive hybridization was performed to isolate DNA from ‘Ca. P. prunorum’ (GSFY2) propagated on Catharanthus roseus. Among 11 new non-ribosomal genetic loci characterized, aceF and pnp genes were selected as targets for detection and sequence typing. PCR primers chosen on aceF and pnp genes allowed to amplify the expected PCR products of 797 bp and 549 bp from the GSFY2 isolate as well as the homologous genetic loci from reference phytoplasma isolates of the group 16SrX. Sequences of aceF PCR products were 13.7% to 15.5% different between ‘Ca. P. prunorum’, ‘Ca. P. pyri’ and ‘Ca. P. mali’, which could be distinguished by specific sequence insertions or deletions. Four different groups of ‘Ca. P. prunorum’ isolates could be detected and discriminated in France according to their aceF sequence. ‘Ca. P. prunorum’ isolates B7, E22, PVCLA8 and PVCLA9, known to be associated with low symptoms and formerly shown to cross protect inoculated trees or peach seedlings against virulent isolates, had the same aceF sequence which was one and two nucleotides different from the aceF sequence of the B6, GSFY, G32 and ECAM200 virulent isolates. A ‘Ca. P. prunorum’ plum isolate detected in Azerbaijan was much more different than the isolates detected in France (10 to 11 nucleotide changes). In addition, the higher variability of aceF by comparison to pnp, makes aceF a better target to monitor the variability of phytoplasmas infecting temperate fruit trees in Europe.