Garden beet (Beta vulgaris L. subsp. esculenta) belongs to family Chenopodiaceae is used as human food or as animal feed. During 2014-16 suspected phytoplasma disease symptoms were observed on garden beet in different areas of Maybod and Abarkooh (Yazd province, Iran). Disease incidence was observed up to 24% and disease symptoms were leaf size reduction, yellowing, phyllody, virescence, proliferation, sterility in the flower, witches’ broom and stunting. For molecular studies total DNA was extracted from 0.2g of midrib tissue of fresh leaves from both symptomatic and symptomless plants. DNA samples were tested for phytoplasma presence by direct and nested PCR using P1/P7, R16mF2/R16mR2 and R16F2n/R16R2 primer pairs. PCR amplicons of ~1.8, ~1.4 and ~1.25 kb respectively were obtained only from symptomatic garden beet plants. Restriction fragment length polymorphism (RFLP) analysis of R16F2n/R16R2 amplicons using KpnI, AluI, HaeIII, HhaI, HpaII, MseI, RsaI, TaqI and HpaI restriction enzymes showed similar patterns belongs to 16SrII group. R16F2n/R16R2 primed amplicons from 4 samples in Maybod and Abarkooh were directly sequenced and sequences showed 100% identity to each other. A consensus sequence of 1,247bp corresponding to the Maybod was deposited in GenBank (GenBank accession: MH428834). Sequence comparison by blast (www.ncbi.nlm.nih.gov) analysis showed the highest sequence identity with phytoplasma members of group 16SrII (peanut witches’ broom). Phylogenetic analysis using neighbor-joining method (MEGA software version 7.0) confirmed that the garden beet witches’broom phytoplasma clustered within the group 16SrII phytoplasma clade, and closer to reference strain of 16SrII-D than to other strains in the same group. Computer-simulated analysis with 17 restriction endonucleases using iPhyClassifier showed that the RFLP pattern derived from the garden beet witches’broom phytoplasma 16S rRNA gene was identical (similarity coefficient 1) to the reference pattern of 16Sr group II, subgroup D (GenBank accession: Y10097). This is the first report of a 16SrII-D phytoplasma associated with garden beet witches’broom disease in Iran.