MicroRNAs (miRNAs) are a class of endogenous RNAs that regulates the gene expression involved in various biological and metabolic processes. Barley is one of the most important cereal crops worldwide and is a model organism for genetic and genomic studies in Triticeae species. However, the miRNA research in barley has lagged behind other model species in grass family. To obtain more information of miRNA genes in barley, we sequenced a small RNA library created from a pool of equal amounts of RNA from four different tissues using Solexa sequencing. In addition to 126 conserved miRNAs (58 families), 133 novel miRNAs belonging to 50 families were identified from this sequence data set. The miRNA* sequences of 15 novel miRNAs were also discovered, suggesting the additional evidence for existence of these miRNAs. qRT-PCR was used to examine the expression pattern of six randomly selected miRNAs. Some miRNAs involved in drought and salt stress response were also identified. Furthermore, the potential targets of these putative miRNAs were predicted using the psRNATarget tools. Our results significantly increased the number of novel miRNAs in barley, which should be useful for further investigation into the biological functions and evolution of miRNAs in barley and other species.