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In this study, heat and relative humidity (HRH) treatment was applied in highland barley for γ-aminobutyric acid (GABA) accumulation. Tibetan highland barley cultivars (25) were selected for comparison and analysis. HRH treatment could accumulate GABA in several hours with low moisture content and high temperature, and the grains were treated for 2.5 h at 65 °C in this study. The GABA content of processed grains under HRH optimal condition ranged from 26.91 to 76.28 mg·100 g−1, which was significantly higher than the initial content (12.78–43.00 mg·100 g−1). The highest GABA accumulation capacity was observed in two-row yellow cultivars (YT1), increasing from 36.52 to 76.28 mg·100 g−1. Correlation analysis showed that the accumulation of GABA after HRH treatment was positively and significantly (p < 0.05) correlated with the contents of protein (0.52), total free amino acids (0.68), threonine (0.53), serine (0.51), glutamate (0.69), glycine (0.49), alanine (0.46), cysteine (0.57), tyrosine (0.50), lysine (0.53), proline (0.40), and glutamate decarboxylase (GAD) activity (0.62), which were closely related to GABA-shunt pathway. The polyamines contents, diamine oxidase (DAO) and polyamine oxidase (PAO) activities, as the substrates and critical enzymes of polyamine degradation pathway, showed no significant correlation with GABA accumulation. The results suggested that the main pathway of GABA accumulation in highland barley under HRH treatment was GABA-shunt pathway.