Elm yellows phytoplasmas (EY) belonging to the 16SrV-A subgroup were recently proposed as a new candidate species ‘Candidatus Phytoplasma ulmi’. These pathogens infect elm trees, causing leaf yellowing and premature drying. In this study, 25 isolates originating from localities in northeast, east and southwest Serbia were characterized by means of RFLP analysis and DNA sequencing of four genomic loci: 16S rRNA, ribosomal protein rpl22-rps3, secY and map. In total, five different genotypes were identified based on collective sequencing of all four genes. Four of these genotypes showed significant nucleotide changes compared with the EY1T reference strain. Phylogeny based on parsimony analyses of ribosomal protein, secY and map genetic loci indicated a single monophyletic origin of EY1T and the new ‘Ca. Phytoplasma ulmi’ strains. Unlike phylogenetic clustering, DNA sequence comparison of EY1T and the novel strains revealed mutations in oligonucleotide signature sequences for all three genes (16S, rpl22-rps3 and secY) used for the characterization and assignment of 16SrV-A phytoplasmas to the ‘Ca. Phytoplasma ulmi’ species in the original description. Based on their high degree of genetic variability, the Serbian strains were assigned to four different subtypes of ‘Ca. Phytoplasma ulmi’ (EY-S1, EY-S2, EY-S3 and EY-S4). New diagnostic enzymes for practical use in ‘Ca. Phytoplasma ulmi’ identification are proposed for the 16S rRNA, ribosomal protein and secY genes. The implications of genetic variability within signature sequences for taxonomy and identification of ‘Ca. Phytoplasma’ species, as well as the importance of geographic variability and number of strains characterized for species description, are discussed.