Lemon balm (Melissa officinalis L.) is one of the most important medicinal plants in the world trade that has many applications in drug and flavoring industrials. In order to evaluate the genetic diversity of different lemon balm populations based on morphological, agronomic and essential oils characters, nine populations from different areas of Iran were collected. In addition, two populations from Germany and Japan were examined. The experiment was carried out based randomized complete block design with three replications. Analysis of variance showed that these populations were significantly different for majority of characters such as plant height, number of tiller, chlorophyll index, leaf width, leaf length, leaf area index, fresh weight, dry weight and dry weight to fresh weight ratio except for number of node and stem diameter. Populations were classified in four groups using cluster analysis based on Ward’s algorithm. High levels of phenotypic variation using Shannon-Weaver diversity index (DI) were found among the countries of origin (DI= 0.95-1.00). Qazvin2 population was more diverse than others (DI = 0.99) whereas populations from Germany and Kurdistan displayed lower diversity indices (DI = 0.96). No clear association was detected between phenotypic diversity and origin of Iranian populations. The results of the character distribution and phenotypic diversity analysis permitted some broad generalization about collection and conservation of lemon balm landraces. This also indicated the presence of important genes for future lemon balm breeding purposes. Despite the limitation in estimating the total genetic variation, the present study indicated that morphological traits were useful for preliminary evaluation and can be used as a general approach for assessing genetic diversity among morphologically distinguishable lemon balm populations.