Aluminum (Al) toxicity is one of the major limiting factors for crop production on acid soils that comprise significant portions of the world's lands. Aluminum resistance in the cereal crop Sorghum bicolor is mainly achieved by Al-activated root apical citrate exudation, which is mediated by the plasma membrane localized citrate efflux transporter encoded by SbMATE. Here we precisely localize tissue- and cell-specific Al toxicity responses as well as SbMATE gene and protein expression in root tips of an Al-resistant near-isogenic line (NIL). We found that Al induced the greatest cell damage and generation of reactive oxygen species specifically in the root distal transition zone (DTZ), a region 1–3 mm behind the root tip where transition from cell division to cell elongation occurs. These findings indicate that the root DTZ is the primary region of root Al stress. Furthermore, Al-induced SbMATE gene and protein expression were specifically localized to the epidermal and outer cortical cell layers of the DTZ in the Al-resistant NIL, and the process was precisely coincident with the time course of Al induction of SbMATE expression and the onset of the recovery of roots from Al-induced damage. These findings show that SbMATE gene and protein expression are induced when and where the root cells experience the greatest Al stress. Hence, Al-resistant sorghum plants have evolved an effective strategy to precisely localize root citrate exudation to the specific site of greatest Al-induced root damage, which minimizes plant carbon loss while maximizing protection of the root cells most susceptible to Al damage.