MicroRNAs (miRNAs) are short, endogenous and non-protein coding RNAs that are 18-26 nucleotides (nt) in length. The miRNAs have been shown to play important regulatory roles in almost all plant processes, including responses to various stresses. These regulatory functions of the miRNAs are to negatively control the protein coding sequences at posttranscriptional level. The mature miRNAs (18-26 nt) are generated from long (50-550 nt) precursor miRNAs (pre-miRNAs). Mostly the pre-miRNAs have one mature miRNA sequence in the stem region, but few have been reported with more than one mature miRNAs. Such miRNAs are called pre-miRNA cluster. In current research, various computational tools were used for the identification and characterization of new conserved pre-miRNA clusters and their targets in barley (Hordeum vulgare L.). Consequently, a total 18 new pre-miRNA clusters were identified from 17 miRNAs families in barley from total 501,838 express sequence tags (ESTs).