The significant loss of sesame harvests in Egypt in recent years can be attributed to phytoplasmas, a class of cell wall-less plant pathogenic bacteria (Mollicutes) that can infect sesame crops. Therefore, before developing and implementing control strategies, it is necessary to characterize the pathogen population. During the 2021 and 2022 growing seasons, samples of sesame leaves displaying phytoplasma symptoms were gathered from six governorate farms. By employing the universal primer pairs P1/P7 and R16F2n/R16R2, nested polymerase chain reaction (PCR) experiments effectively identified phytoplasma, and the resultant product of about 1800 bp was amplified. Its infection rate was highest in Giza, at 29% and 25%, respectively, and lowest in Ismailia, at 16.2% and 15.7%, respectively. Phylogenetic analysis, sequencing restriction profiling, and nested polymerase chain reaction (PCR) tests based molecular techniques demonstrated that our Egyptian isolate, accession number OR447565.1, is closely related to the 16SrII (Peanut WB group) and that it is very closely (98.86%) identified with isolates of MN565885.1 and OP793488.1 from Iran and India, respectively, which belong to the same subgroup. In addition, the comparison showed that the Egyptian isolate (OR447565.1) shared 16SrVI (Clover proliferation group). A discernible degradation in the tissue and cell ultrastructure of phytoplasma-infected plants was found by Transmission Electron microscopy (TEM) analysis. Pleomorphic phytoplasma units were seen. Attached to the sieve elements plasma membrane.