We aim to identify conserved and dehydration responsive microRNAs (miRNAs) in Hordeum vulgare (barley). A total of 28 new barley miRNAs belonging to 18 distinct miRNA families were identified. Detailed nucleotide analyses revealed that barley pre-miRNAs are in the range of 46-114 nucleotides with average of 77.14. Using 28 newly detected miRNAs as queries, 445 potential target mRNAs were predicted. The predicted miRNAs were differentially expressed and some of them behaved similarly in leaf and root tissues upon stress treatment. Hvu-MIR156, Hvu-MIR166, Hvu-MIR171, and Hvu-MIR408 were detected as dehydration stress-responsive barley miRNAs. To discover target transcripts of barley miRNAs a modified 5' RLM-RACE was performed and seven cleaved miRNA transcripts were retrieved from drought stressed leaf samples. In silico analysis indicated 15 potential EST targets. Measurement of expression levels showed a positive correlation between levels of miRNA expression and suppression of their target mRNA transcripts in dehydration-stress-treated barley.