Abstract: A new disease of hemp plants characterized by witches'-brooms and stunting symptoms was appeared in Yazd province in the centre of Iran. Phytoplasma infections were detected in symptomatic hemps by polymerase chain reaction (PCR) amplifications using phytoplasma universal primer pair P1/P7 followed by R16F2n/R16R2 in nested PCR. Restriction fragments length polymorphism (RFLP) analysis results using CfoI restriction enzyme confirmed that the hemp witches'-broom phytoplasma isolates are related to stolbur group. Also, virtual RFLP analysis revealed that hemp witches'-broom phyutoplasmas in Iran are different from members of other 16SrXII subgroups. Sequence analysis of partial 16S rRNA gene indicated that the phytoplasmas associated with hemp in Iran shared high similarity with ‘Candidatus Phytoplasma solani’ and other phytoplasmas related to stolbur group. This research is the first report of hemp infection with phytoplasmas related to stolbur group.