Background /Methodology: Taking advantage of a newly available barley (Hordeum vulgare) genome sequence assembly and increasing number of miRNAs identified in other plant species, we carried out computational analyses to identify and characterize miRNAs conserved in barley. We investigate the locations of miRNAs on the barley genome assembly and provide annotation of the functions of their predicted target genes. We compare our results to previous miRNA studies and publicly available barley small RNA libraries. Results: 116 mature miRNA sequences from 60 miRNA families have been found in the barley genome assembly by our miRNA identification pipeline. Closely related cereal crops contain most of the miRNA families that we found in the barley genome assembly. Most miRNA genes were located in intergenic regions or introns. Among the 116 mature miRNAs predicted, 80 have been reported in previous barley miRNA studies. Eight mature miRNA sequences have never reported in the previous barley miRNA studies. Conclusions: This in silico study has provided updated information in characterizing plant miRNAs in barley. The identified miRNA and precursor sequences, their genomic locations as well as predicted target transcripts will serve as valuable resources for future studies.