Essential oils are generally produced to confer the protection of medicinal plants against several natural enemies. Variations of chemical and physical environmental factors exert significant influences on plant development. They hence may affect the quality and quantity of volatile organic metabolites of interest and, therefore, the economic applications of essential oils. This research focused on the effects of the harvest region on the production and analytes present in Tunisian Pimpinella lutea Desf. Apiaceae that were collected in three different growing environments (North and South Bizerta and Tabarka). Essential oils extracted from a variety of genotypes were analyzed, for the first time, using gas chromatography and mass spectrometry (GC/FID and GC/MS). The determination of the percentage of essential oil components allowed the recognition of three chemotypes: α-trans-Bergamotene quantified at a percentage of 18.1% in North Bizerta (NBEO), muurola-4,10(14)-dien-1-β-ol identified in South Bizerta (10.1%, SBEO) and acora-3,7(14)-dien present in a high level of 29.1% in Tabarka population (TEO). The richness of different populations in sesquiterpenes (60.2–78.1%) suggests that Pimpinella lutea Desf. may be used in different industrial segments.