Mexican oregano (Lippia graveolens), belonging to the Verbenaceae family, is an aromatic and perennial herb that produces an essential oil rich in the monoterpenes thymol and carvacrol, widely utilized in various industries. Endemic to Mexico, it predominantly thrives in arid and semi-arid regions, typically displaying notable drought tolerance. However, previous studies reveal that irrigation frequency significantly influences biomass production, prompting the need for further improvement in drought tolerance in this species, especially when considering future climate change scenarios. This study employed chemical mutagenesis with ethyl methanesulfonate (EMS) to create new genetic variants through induced mutations. Seeds of L. graveolens underwent EMS treatment at varying concentrations (0.1 and 0.2%) and exposure times (1, 3 and 6 hours), and then aseptically germinated on MS medium. Nodal segments from resulting seedlings were used as explants for multiple shoot proliferation using 50 g·L-1 of polyethylene glycol (PEG) as a selective agent for drought tolerance, where non-mutagenized plants displayed severely inhibited development and necrosis. Twenty-five putative mutants tolerant to osmotic stress were recovered, and some of them showed evident morphological alterations and significant changes in the content of phenols and flavonoids, compounds associated with responses to stress. These results highlight the effectiveness of chemical mutagenesis as a strategy for genetically enhancing drought tolerance in Mexican oregano.