The immunodominant membrane protein Imp of several phytoplasmas within the ‘Candidatus Phytoplasma aurantifolia’ (16Sr-II) group was investigated. Eighteen isolates from Iran (11), East Asia (5), Africa (1) and Australia (1) clustered into three phylogenetic subgroups (A, B and C) based on the 16S rDNA and imp genes, regardless of geographic origin. The imp gene sequences were variable, with more non-synonymous than synonymous mutations (68 vs 20, respectively), even though many of the non-synonymous ones (75%) produced conservative amino acid replacements. Eight codon sites on the extracellular region of the protein were under positive selection, with most of them (75%) coding for non-conservative amino acid substitutions. Full-length (21 kDa) and truncated (16 kDa) Imp proteins of two economically important Iranian phytoplasmas [lime witches’ broom (LWB) and alfalfa witches’ broom (AlWB-F)] were expressed as His-tagged recombinant proteins in Escherichia coli. An antiserum raised against full-length recombinant LWB Imp reacted in western blots with membrane proteins extracted from LWB-infected periwinkle and lime, indicating that Imp (19 kDa) is expressed in infected plants and is a membrane-associated protein. The same polyclonal antibody also detected native Imp in proteins from periwinkles infected by phytoplasmas closely related to LWB (subgroup C) only, confirming phylogenetic clustering based on 16S rDNA and imp genes. Imp proteins of LWB and AlWB-F isolates were also recognized by an antiserum raised against an enriched preparation of AlWB-F phytoplasma cells, demonstrating the antigenic properties of this protein.