Almond witches' broom (AlWB) is a destructive phytoplasma disease in Iran and Lebanon. In this study 50 almond trees with suspected symptoms (very mild to severe) were sampled from almond groves of Fars and Chaharmahal-Bakhtiari provinces during spring and summer of 2016. Total DNA was extracted from midribs of leaves by standard CTAB method. Detection of phytoplasma was performed by nested PCR analysis using primer pairs P1/P7 and R16mF2/R16mR1. Fragments with expected size were amplified from 2 (out of 32) and 13 (out of 18) plant samples from Chaharmahal-Bakhtiari and Fars provinces, respectively. Based on virtual RFLP patterns, sequence identity and phylogenetic analyses, phytoplasmas detected in 2 and 11 samples from, respectively, Chaharmahal-Bakhtiari and Fars provinces, belonged to the subgroup 16SrIX-B ('Ca. P. phoenicium'). Phytoplasmas detected in the other 2 samples from Fars province belonged to the subgroup 16SrIX-C. The 16S rRNA gene sequence of all phytoplasmas of 16SrIX-B had the highest similarity (99.9%) with that of the reference AlWB phytoplasma strain A4 from Lebanon. The sequence of rplV-rpsC genes from phytoplsams of this study was also determined. Pairwise nucleotide sequence identity of rplV-rpsC among phytoplasmas of 16SrIX-B ranged between 99.7-100% and was 100% between phytoplasmas of 16SrIX-C. The average rplV-rpsC nucleotide sequence identity between phytoplasmas of 16SrIX-B and IX-C was 90.9%. BLAST analysis revealed that phytoplasmas of 16SrIX-B in this study had the highest rplV-rpsC similarity, ranging between 98.9 and 99.2, with that of AlWB phytoplasma strain A112 from Lebanon. This is the first report of sequencing of rplV-rpsC genes from Iranian phytoplasma strains of AlWB belonging to 16SrIX-B. BLAST analysis also showed that the other the 2 AlWB phytoplasmas of 16SrIX-C (this study) had the highest rplV-rpsC nucleotide sequence similarity (99.3%) with its counterpart in Knautia arvensis phyllody phytoplasma belonging to 16SrIX-C. The results of this study revealed that phytoplasma strains of 16SrIX-B were more prevalent than phytoplasmas of 16SrIX-C in association with AlWB disease in Fars province. Also we report, for the first time, the restricted distribution of phytoplasmas belonging to 16SrIX-B associated with AlWB in Chaharmahal-Bakhtiari. This study also recognized rplV-rpsC as a powerful genetic marker for finer differentiation of AlWB phytoplasma strains in Iran and Lebanon.