Plant root exudates are involved in nutrient acquisition, microbial partnerships, and inter-organism signaling. Yet, little is known about the genetic and environmental drivers of root exudate variation at large geographical scales, which may help understand evolutionary trajectories of plants in heterogeneous environments. We quantified natural variation in chemical composition of Arabidopsis thaliana root exudates in 105 Iberian accessions. We identified up to 373 putative compounds using ultra-high performance liquid chromatography coupled with mass spectrometry. We estimated broad-sense heritability of compounds and conducted a genome-wide association (GWA) study. We associated variation in root exudates to variation in geographic, environmental, life history, and genetic attributes of Iberian accessions. Only 25 of 373 compounds exhibited broad-sense heritability values significantly different from zero. GWA analysis identified polymorphisms associated to 12 root exudate compounds and 26 known genes involved in metabolism, defense, signaling, and nutrient transport. The genetic structure influenced root exudate composition involving terpenoids. We detected five terpenoids related to plant defense significantly varying in mean abundances in two genetic clusters. Our study provides first insights into the extent of root exudate natural variation at a regional scale depicting a diversified evolutionary trajectory among A. thaliana genetic clusters chiefly mediated by terpenoid composition.