The variation of the essential-oil composition among 14 Tunisian natural populations of Thymus algeriensis Boiss. et Reut. (=Thymus hirtus Willd. ssp. algeriensis Boiss. et Reut.) was assessed by GC (RI) and GC/MS. The populations were collected from different geographical regions belonging to the sub-humid, upper semi-arid, mean semi-arid, lower semi-arid, and upper-arid bioclimates. A total of 47 constituents, representing 81.0 to 96.5% of the total oil, were identified. The main volatiles at the species level were 1,8-cineole (17.7%), alpha-pinene (15.5%), and camphor (8.2%). A high variation among populations for the majority of the compounds was shown. Camphor (0.2-14.0%), linalool (0.2-22.4%), borneol (<0.01-24.3%), caryophyllene oxide (<0.01-18.8%), thymol (<0.01-54.9%), gamma-terpinene (0.4-6.5%), alpha-copaene (0.4-7.6%), linalyl acetate (<0.01-6.4%), and methyl eugenol (<0.01-6.9%) were the main constituents differentiating the populations. The chemical differentiation among populations, assessed by principal component analysis (PCA) and a UPGMA (unweighted pair-group method with averaging) cluster analysis performed on all populations and compounds, was high. Six chemotypes according to the main compounds have been distinguished, i.e., caryophyllene oxide/1,8-cineole/alpha-pinene, 1,8-cineole/alpha-pinene, 1,8-cineole/alpha-pinene/camphor, borneol/1,8-cineole/alpha-pinene, linalool, and thymol chemotypes. The spatial chemotype distribution was linked to the geographic distance among populations rather than to bioclimates, indicating that local selective environmental factors act on the chemotype diversity. The high chemical variation among populations according to their geographical and bioclimatic distribution imposes that conservation strategies of populations should be made appropriately, taking into account these factors. The in situ and ex situ conservation strategies should concern all populations representing the different chemotypes.