‘Candidatus Phytoplasma ulmi’ (16SrV-A) is the causal agent of elm yellows (EY), a lethal and/or decline disease of several Ulmus (elm) species and hybrids in North America and Europe. In this study, field observations and PCR assays were used to detect phytoplasma infections in diseased U. minor, U. pumila and U. glabra trees in southern Italy. Also, a multigene sequence analysis employing various less conserved genes was carried out to explore the genetic variation in detected strains. All the symptomatic elm trees tested were infected with ‘Ca. Phytoplasma ulmi’. No other phytoplasmas or variants could be detected. Although ‘Ca. Phytoplasma ulmi’ was already known to occur in southern Italy on European field elm, the current work expands the information on the presence, disease incidence and severity, plant host range and molecular aspects of EY phytoplasma strains occurring in southern Italy. In addition, this is the first report from Italy on the molecular characterization of EY phytoplasma strains through map, imp and groEL gene sequence and phylogenetic analyses. Among the newly detected EY phytoplasma strains, some proved distantly related to each other and to other previously characterized EY phytoplasma strains within the genes examined. This implies the presence of distinct taxonomic entities within the material examined. The occurrence of different strains was not linked to the biological traits and geographical distribution. However, the data obtained may provide a basis for further studies aimed at elucidating several other unknown aspects of the EY agent, knowledge of which is essential for effective disease management and control strategies. The results of the current work also show that the EY phytoplasma is particularly widespread in southern Italy and is of considerable economic and ecological relevance.