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The chemical compositions of the essential oils of seven natural populations of Seseli rigidum were analyzed. The essential-oil yield ranged from 0.16 to 2.09%. Analysis of variance (ANOVA) revealed that there were no statistically significant differences in the mean essential-oil yields between the populations, and no significant influence of the climate or soil type on the oil yield was observed. In all 67 analyzed samples, the polyacetylene falcarinol was the main compound, followed by octanal, methyl linoleate, -muurolene, 3-butylphthalide, falcarinone, muurola-4,10(14)-dien-1-ol, -sesquiphellandrene, salvial-4(14)-en-1-one, -amorphene, spathulenol, and isospathulenol. The principal component analysis (PCA), the canonical discriminant analysis (CDA), and the cluster analysis (CA) revealed differentiation between the populations based on the climate. Three groups of populations were formed; the first group was composed of samples growing in regions with a humid climate, with oils having high falcarinol and low sesquiterpene contents, and the second and third groups comprised samples exposed to semi-arid climate, with oils characterized by a lower falcarinol and higher -muurolene, -amorphene, -sesquiphellandrene, and salvial-4(14)-en-1-one contents. The semi-arid populations were divided into two groups, which were distinguished based on the oil contents of sesquiterpenes, falcarinone, and 3-butylphthalide. On the other hand, no clear separation between populations based on the different soil types could be observed.