This study focused on evaluating the genetic diversity among ‘Candidatus Phytoplasma mali’ (‘Ca. P. mali’) populations in orchards of north-western Italy, where apple proliferation (AP) disease is widespread and induces severe economic losses. ‘Ca. P. mali’ was detected through restriction fragment length polymorphism (RFLP) analysis of PCR-amplified 16S rDNA in 101 of 114 samples examined. Collective RFLP patterns, obtained by restriction analyses of four amplified genomic segments (16S/23S rDNA, PR-1, PR-2 and PR-3 non-ribosomal region, ribosomal protein genes rplV-rpsC and secY gene), revealed the presence of 12 distinct genetic lineages among 60 selected representative ‘Ca. P. mali’ isolates, underscoring an unexpected high degree of genetic heterogeneity among AP phytoplasma populations in north-western Italy. Prevalence of distinct genetic lineages in diverse geographic regions opens new interesting avenues for studying the epidemiology of AP disease. Furthermore, lineage-specific molecular markers identified in this work could be useful for investigating the biological life cycle of ‘Ca. P. mali’.