The subgroup classification of phytoplasmas in 16S rRNA group 16SrV (elm yellows phytoplasma group) was revised and extended on the basis of enzymatic restriction fragment length polymorphism (RFLP) analysis of ribosomal (r) DNA and analysis of putative restriction sites in nucleotide sequences. A 1.85 kbp fragment of the rRNA operon from flavescence dorée (FD) phytoplasma strain FD70 from France was amplified and cloned, and its nucleotide sequence determined (GenBank acc. no. AF176319). Placement of FD70 in subgroup V-C was verified by analysis of amplified DNA and of the cloned sequence. Hemp dogbane phytoplasma HD1 (AF122912), a member of subgroup V-C, was distinguished from other subgroup V-C phytoplasmas by putative restriction site differences in the 16S-23S rRNA spacer region. A previously published FD phytoplasma sequence (GenBank accession no. X76560) differed from FD70 sequence AF176319 by at least eight nucleotide substitutions and differences in putative restriction sites. The X76560 FD phytoplasma was classified in a new subgroup (V-D). Based on analyses of 16S rDNA GenBank sequence Y16395, Rubus stunt phytoplasma was classified in new subgroup V-E. The revised classification was supported by sequence similarities, group 16SrV-characteristic sequences, and a phylogenetic tree constructed on the basis of 16S rDNA sequences.