Rapeseed (Brassica napus) is an economically important oil crop grown worldwide and in Iran. In April 2018, several rapeseed plants with typical symptoms of phytoplasms diseases were observed in the rapeseed farms of Hormozgan province, Iran. The symptomatic plants showed witches broom, plant stunting (shortened internodes) and little leaves. To identify association of phytoplasma with the symptomatic plants, the total genomic DNA was extracted from 0.2 g midribs of five symptomatic and two asymptomatic plants using modified cetyltrimethylammonium bromide (CTAB) method. The samples were analyzed for phytoplasma DNA by direct PCR using the phytoplasma universal primer pair P1 / P7 and nested PCR using primers P1 / P7 (first round) followed by R16F2n / R16R2 (second round). Amplicons of the expected sizes (1800 and 1250 bp) were amplified with direct and nested-PCR in all symptomatic plants but not from symptomless plant or sterile distilled water as negative controls. A positive nested-PCR product was directly sequenced and edited. BLAST search using sequenced fragment showed that the phytoplasma associated with rapeseed witches' broom (RsWB) had 100 % nucleotide identity with those of phytoplasma members of group 16SrVI (KX773529, KP864671 and AM260486), Candidatus Phytoplasma trifolii. There are several reports regarding association of phytoplasmas diseases with rapeseed (such as rapeseed phyllody phytoplasma) in Iran and other countries, which all are closer to the members of the aster yellows group (16SrI). To our knowledge, this is the first report of natural infection of rapeseed by a 16SrVI phytoplasma in Iran and most probably in the world.