Soil mechanical impedance precludes root penetration, confining root system development to shallow soil horizons where mobile nutrients are scarce. Using a two-phase-agar system, we characterized Arabidopsis thaliana responses to low and high mechanical impedance at three root penetration stages. We found that seedlings whose roots fail to penetrate agar barriers show a significant reduction in leaf area, root length and elongation zone and an increment in root diameter, while those capable of penetrating show only minor morphological effects. Analyses using different auxin-responsive reporter lines, exogenous auxins and inhibitor treatments suggest that auxin responsiveness and PIN-mediated auxin distribution play an important role in regulating root responses to mechanical impedance. The assessment of 21 Arabidopsis accessions revealed that primary root penetrability (PRP) varies widely among accessions. To search for quantitative trait loci (QTLs) associated to root system penetrability, we evaluated a recombinant inbred population (RIL) derived from Landsberg erecta (Ler-0, with a high PRP) and Shahdara (Sha, with a low PRP) accessions. QTL analysis revealed a major-effect QTL localized in chromosome 3 (q-RPI3), which accounted for 29.98% (LOD = 8.82) of the total phenotypic variation. Employing an introgression line (IL-321), with a homozygous q-RPI3 region from Sha in the Ler-0 genetic background, we demonstrated that q-RPI3 plays a crucial role in root penetrability. This multiscale study revels new insights into root plasticity during the penetration process in hard agar layers, natural variation and genetic architecture behind primary root penetrability in Arabidopsis.