Witches' broom of alfalfa (Medicago sativa L.) is a serious disease caused by a phytoplasma in the Al-Batinah, Al-Sharqiya, Al-Dhahera, and interior regions of the Sultanate of Oman. Disease incidence was recorded to be 95% in 6 year-old alfalfa crop during field survey. Phytoplasma bodies were detected by 4,6 diamidino-2-phenylindole (DAPI) staining in alfalfa plants showing witches' broom symptoms. Results obtained from transmission electron microscopy revealed the presence of numerous phytoplasma cells in phloem sieve-tube members in diseased plants but not in healthy tissues. Total nucleic acid isolated from symptomatic alfalfa was used as template to amplify 16S-23S ribosomal DNA and spacer region by polymerase chain reaction (PCR) using two pairs of universal primers as direct (P1/P7) and nested (R16F2n/ R16R2). The PCR amplifications yielded 1.8 and 1.2 kb fragment from direct and nested PCR respectively from DNA of symptomatic alfalfa, Restriction fragment length polymorphism (RFLP) profiles of direct PCR products amplified from symptomatic alfalfa and 19 phytoplasmas from different groups used as positive control with Tsp5091, Rsal, Tagl, Trugl, and Hpall restriction enzymes clearly indicated that alfalfa phytoplasma is different from all other phytoplasmas employed in this study belonging to subgroup 16Srll, except tomato big bud (TBB) phytoplasma from Australia, and could be therefore classified in TBB subgroup, 16Srll-D. The P1/P7 PCR product was sequenced after cloning and yielded 1690 bp product. The alfalfa witches' broom (AlfWB) phytoplasmsa showed >98% similarity with papaya yellow crinkle (Papaya YC) phytoplasma from New Zealand in the homology search and <98% similarity with LWB phytoplasma. These finding were supported by phylogenetic analysis that showed AllWB clustering with FBP group (16Srll) and PapayaYC belonging to the same group. On the basis of results obtained from restriction fragment length polymorphism and nucleotide sequence analyses of 16S rDNA, the AlfWB phytoplasma was classified in FBP group (16Srll), designated subgroup II-D.