Mentha aquatica L. (Lamiaceae) is found in different parts of Iran. Its essential oil and preparations regulate bile function and are used as a stomach tonic and disinfectant. This study investigates the morphological and essential oil diversity of M. aquatica populations from the Hyrcanian hotspot of Iran. Plant samples were collected from Gilan, Golestan, and Mazandaran provinces in the Caspian Region for analysis. The results showed significant differences among the studied ecotypes for the stem diameter, collar diameter, number of inflorescences, length and width of inflorescence, sepal diameter, sepal length, and secondary stem length number. Principal component analysis showed that the first seven principal components explained 90.6% of the total variation. Moreover, essential oil concentration varied widely from 1.13% for a sample from Behshahr-Mazandaran, down to 0.27% for one from Abbas abad-Mazandaran. GC–MS analysis identified 29 constituents that accounted for 91% of the total essential oil. The main components of the essential oil were menthofuran (13.21–52.46%), 1,8-cineole (12.42–25.55%), (E)-caryophyllene (3.18–15.43%), viridiflorol (1.04–11.16%), germacrene D (1.70–8.29%), caryophyllene oxide (0.51–4.96%), neryl acetate (1.11–4.95%), p-cymene (1.55–4.77%), and β-pinene (1.7–3.45%). Overall, meaningful diversity was recorded among the populations; Rahimabad-Gilan and Behshahr-Mazandaran would be reliable selections for the food and pharmaceutical industries due to their higher yields and content of α-pinene, 1, 8-cineole, menthofuran, viridiflorol, and β-caryophyllene. Further evaluation of populations from diverse habitats is needed to guide future breeding programs.