Late embryogenesis abundant (LEA) proteins are thought to protect against water stress in plants. Characteristics of sequence and expression of barley gene HVA1, a member of LEA group 3 protein, were investigated in hulless barley (Hordeum vulgare ssp. vulgare), associated with phenotypically diverse drought-tolerant genotypes. Sensitive and tolerant genotypes were identified from Tibetan populations of cultivated hulless barley, based on scores of water loss rate (WLR), maldondialdehyde (MDA), and proline content. The results indicated that lower MDA contents, lower scores of WLR, and higher proline contents were associated with drought-tolerant genotypes in hulless barley. Notably, differential trends of expression patterns were detected among the selected contrasting genotypes, depending on the duration of dehydration stress. The HVA1 gene tended to respond earlier in the tolerance (after 2 h) compared with sensitive genotypes (after 4 h). Results of quantitative real-time PCR indicated that the relative level of HVA1 expression was always higher in tolerant genotypes, rapidly increasing at the earlier stages (after 2–4 h of dehydration). However, HVA1 expressions of sensitive genotypes had a fast increase from 8 to 12 h of stress. Variable numbers of the 11-amino-acid-motif in LEA3 proteins were not consistent with the lines of drought resistance in hulless barley. Molecular characteristic of LEA3 protein in tolerant lines existed in the consistency of Gln₃₂, Arg₃₃, and Ala₁₉₅ in Tibetan hulless barley. The present study may indicate that the differential HVA1 gene has a functional role in the dehydration tolerance in hulless barley. The authors suggested that the observed variability in sequence and expression of HVA1 could be related to the diverse drought-tolerant genotypes in crops.