MicroRNAs are small, non-protein-coding RNAs playing regulatory functions in many organisms. Using computational approaches 26 new Brachypodium distachyon miRNAs belonging to 19 miRNA families were identified in expressed sequence tags (EST) and genomic survey sequence databases. EST revealed that predicted miRNAs are expressed in B. distachyon. Detailed nucleotide analyses showed that pre-miRNAs in B. distachyon are in the range of 63-180 nucleotides. Mature miRNAs located in the different positions of precursor RNAs are varied from 19 to 24 nucleotides in length. Quantifying RNAs using realtime PCR (qRT-PCR) analyses validated expression level differences of selected B. distachyon miRNAs. In this study, we detected that the expression level of some of the predicted miRNAs are distinct and some of them are similar in the leaf tissues. In addition, using these miRNAs as queries 27 potential target mRNAs were predicted in B. distachyon NCBI EST database and 246 target mRNA were predicted in NCBI protein-coding nucleotide (mRNA) database of all plant species. The majority of the target mRNAs encode transcription factors regulating plant development, morphology and flowering time. Other newly identified miRNAs target the mRNAs involving metabolic processes, signal transduction and stress response.