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The medicinal herb, lemon balm ( Melissa officinalis L.), which is high in rosmarinic acid (RA), has well-known therapeutic value. The goals of this study were to investigate the effects of methyl jasmonate (MeJA) on RA content, total phenolic content (TPC), and total flavonoid content (TFC), as well as changes in expression of their biosynthesis-related key genes ( MoPAL , Mo4CL , and MoRAS ) in Iranian lemon balm ecotypes, as first reported. Our results revealed that MeJA doses significantly increase the RA content, TPC, and TFC in both ecotypes compared with the control samples. Additionally, the higher expression levels of MoPAL , Mo4CL , and MoRAS following treatment were linked to RA accumulation in all treatments for both Iranian lemon balm ecotypes. After 24 h of exposure to 150 µM MeJA concentration, HPLC analysis showed that MeJA significantly increased RA content in Esfahan and Ilam ecotypes, which was about 4.18- and 7.43-fold higher than untreated plants. Our findings suggested that MeJA has a considerable influence on RA, TPC, and TFC accumulation in MeJA-treated Iranian M. officinalis , which might be the result of gene activation from the phenylpropanoid pathway. As a result of our findings, we now have a better understanding of the molecular processes behind RA production in lemon balm plants.