Mapping of quantitative trait genes (QTGs) associated with drought related traits is essential for improving drought tolerance in crop species. In silico identification of candidate genes relies on annotation of critical QTGs to a variety of web resource-based datasets. The barley reference sequence was employed to map QTGs significantly associated with the proline accumulation and osmotic potential. Annotation of the critical QTGs contigs to the NCBI protein database identified 72 gene orthologs located on chromosomes 1H, 2H, and 7H, from which seven genes were identified as candidates. Expression analysis of all seven candidate genes revealed differential expression pattern between plants grown under well-watered conditions and drought-stress. The results represent a successful and highly powerful implementation of genome-wide scanning approach based on in silico mapping of QTGs to identify gene clusters having a common transcript pattern with similar function.