Plants are usually exposed to several types of abiotic stress in regular field conditions. The lipid profile of barley homozygous lines exposed to drought, heat, salinity, and their combinations, was investigated in the present study. Free fatty acids, free sterols, and diacylglycerols were the most abundant classes (∼8.0% of plant material). The genetic background significantly impacted the lipid composition rather than the treatments, and diacylglycerols were the only lipid class affected by salinity (1.84 mg/100 mg plant tissue; ∼33% reduction). However, the genotype × treatment interaction analysis revealed that the lipid and sterol compositions depended on both genotype and environment. Our results suggest that inborn stress tolerance in barley is manifested by enhanced accumulation of most lipids, mainly sterols, especially in heat/drought-stressed plants. In addition, expression of the LTP2 gene may be indirectly involved in the abiotic stress reaction of barley by mediating intracellular transport of some lipid classes.