<p>Herbal spices and human health are inextricably intertwined, and they can be utilized as medicines around the world. Global demand for ajwain production increased tremendously due to its diverse utility; it can improve the flavour and promote human health by treating several ailments. In recent years, researchers have become increasingly interested in their medical and therapeutic applications. Ajwain suffers from a narrow genetic base, which limits progress through conventional breeding. Therefore, the primary objective of this study was to evaluate the mutagenic efficacy of two chemical mutagens, ethyl methanesulphonate (EMS) and hydrazine hydrate (HZ), at varying doses for generating genetic variability and selecting improved putative mutant lines with enhanced agronomic traits in ajwain. Two high-yielding putative mutants were isolated as a consequence of a thorough phenotypic selection process that emphasized variables such as plant height (cm), days to flowering, days to maturity, fertile branches per plant, umbels per plant, seeds per umbel, and 1000-seed weight (g) and seed yield. The results showed that the studied parameters exhibited diminution and also amplification at higher treatments compared to the lower treatments. The maximum increase in mean seed yield was recorded as 3.80&#xa0;g in 0.2% EMS and 3.50&#xa0;g in 0.01% HZ, respectively. The significant difference between the means of mutagenized and control populations was analysed by using the Duncan multiple range test (DMRT). Mutagen induced genetic divergence was estimated in ajwain developed by the treatments of EMS and HZ using hierarchical cluster analysis. Four clusters were created for EMS treatments, and in the case of HZ treatments, three clusters were created. Mutant plants with increased yields can be effectively utilized in future breeding programmes to boost the variability of existing varieties.</p>