Valeriana jatamansi is an important temperate herb, which is used for pharmaceutical and essential oil industries. In India, this species is now at the verge of extinction due to the over-exploitation of its rhizomes from its natural habitat. It is hypothesized that the variations in bioactive compounds in its essential oil are very high among the wild populations as well as cultivated sources. Thus, the study was conducted to evaluate the chemical profiling of essential oil of four wild populations (Rupena, Kugti, Garola, Khani) and two cultivated sources (CSIR-IHBT, Saloonie), which were distilled at three consecutive days. The variation in oil concentration in root/rhizome was found significant (P ≤ 0.05), and the maximum value (0.35%) was registered with the population collected from Kugti and Khani. In essential oil, irrespective of population and distillation day, patchouli alcohol was the major compound, which ranged from 19 to 63.1%. The maximum value (63.1%) was recorded with the essential oil obtained from Garola's population and distilled on the first day. The percentage of seychellene was abruptly increased with subsequent days of extraction in all the populations. The multivariate analysis revealed that the essential oil profiles of Rupena, Kugti, Garola, and CSIR-IHBT populations were found similar during the first day of distillation. However, during the second day Rupena, Kugti, Khani, and CSIR-IHBT come under the same ellipse of 0.95% coefficient. The results suggest that the population of Kugti is superior in terms of oil concentration (0.35%) with a higher proportion of patchouli alcohol (63% on the first day). Thus, repeated distillation is recommended for higher recovery of essential oil. Moreover, repeated distillation can be used to attain V. jatamansi essential oil with differential and perhaps targeted definite chemical profile.