Pepper Capsicum annuum is one of the important vegetable crops in Iran, especially north of Iran. Various symptoms of stolbur, including limited growth, small and chlorotic leaves, spoon-shaped leaflets, and sterility or fruit alterations, were detected in samples collected from the pepper field in Qazvin province. DNA was extracted from midribs and petioles of pepper leaves using CTAB-based methods. The phytoplasma in all symptomatic pepper plant parts was detected by direct and nested polymerase chain reactions (PCR) using primer pairs P1/P7 and R16F2n/R16R2. The 16S rDNA sequences of phytoplasma isolate were deposited in GenBank (MN877916). Based on phylogenetic studies of the 16S rDNA region, the results of enzymatic digestion of the fragment obtained by amplification with R16F2n/R16R2 primer and virtual RFLP, phytoplasma agent associated with stolbur pepper disease was detected to belong to 16SrXII group and 16XII-A subgroup. According to our knowledge, this is the first report of pepper stolbur disease in Iran.