Ocimum viride is known for its diverse medicinal uses. However, most previous studies have been conducted on phytochemical assessment, but intra-specific chemical variations are rarely reported. Chemical variations among seven genotypes of O. viride were studied over three consecutive years in the experimental farm in CSIR-CIMAP, Lucknow. The essential oils obtained by hydro-distillation from the aerial parts of O. viride genotypes were subjected to GC, enantiomeric GC, and GC/MS analyses for their chemical characterization. GC-FID analysis revealed maximum proportions of eugenol (37.8 %–45.8 %) along with monoterpenes such as (Z)-β-ocimene (9.3 %–29.5 %). The sesquiterpenoids identified were β-caryophyllene (3.1 %–4.6 %), α-trans-bergamotene (5.5 %–7.4 %) and germacrene D (5.9 %–10.6 %). Chiral separation of germacrene D enantiomers [(+)– and (−)–form] were achieved using a cyclodextrin-based capillary column in each genotype. (−)–Germacrene D was found to be the most predominant enantiomer in all genotypes. Genotype 133 was recorded with the highest enantiomeric excess (≥ 99 %) for (−)–germacrene D. Besides, genotype 131 contained high monoterpenoids. Major chemical constituents such as eugenol were isolated through column chromatography, and structure elucidation was carried out using 1D and 2D NMR experiments. The thermo-gravimetric analysis (TGA) showed the stability pattern of extracted essential oils and revealed the complete volatile degradation ranged from 160 °C–200 °C. The statistical analysis was performed to select the best genotypes. The study revealed the best genotypes based on the correlation between constituents × years (C × Y) interaction. These analyses helped better estimate essential oil yield analysis, chemical diversity, and herb yield analysis on a field experiment basis. In conclusion, genotypes 129 and 132 had the highest essential oil output and eugenol content (%). Eugenol, a marker chemical ingredient, was reported with the highest proportions in genotype 132 at the full bloom stage.