Aluminium is the third most abundant element in the Earth’s crust after oxygen and silicon. In neutral or alkaline soils it occurs mainly in stable mineral forms that are not harmful to plants. Under acidic conditions, however, aluminium becomes soluble and forms highly reactive and phytotoxic Al³⁺ ions. As more than half of the world’s soils are acidic, and soil acidification is further intensified by industrial activity and the use of ammonium-based fertilisers, aluminium toxicity represents a major limitation to agricultural productivity. Plants have evolved several mechanisms to mitigate aluminium toxicity. One important strategy involves the secretion of organic acid anions from root cells into the rhizosphere, where they chelate toxic Al³⁺ ions. In addition, aluminium can be detoxified through sequestration into the vacuole. These processes are largely regulated by transcription factors such as STOP1 (Sensitive To Proton Rhizotoxicity 1), which control the expression of genes associated with aluminium tolerance. Among these genes, the ABC-family transporter ALS1 is thought to play a key role in the vacuolar transport of aluminium–organic acid complexes. This study focuses on aluminium stress responses in barley (Hordeum vulgare), the fourth most important cereal crop worldwide, primarily cultivated for animal feed and considered one of the most aluminium-sensitive crop species. We present preliminary results suggesting the involvement of selected barley genes in aluminium stress responses. The analyses were performed using mutant lines identified through a TILLING approach in the HorTILLUs population.