MicroRNAs (miRNA) are a class of small, endogenous RNAs that play a negative regulatory role in various developmental and metabolic processes of plants. Wild barley (Hordeum vulgare subsp. spontaneum), as the progenitor of cultivated barley (Hordeum vulgare subsp. vulgare), has served as a valuable germplasm resource for barley genetic improvement. To survey miRNAs in wild barley, we sequenced the small RNA library prepared from wild barley using the Illumina deep sequencing technology. A total of 70 known miRNAs and 18 putative novel miRNAs were identified. Sequence analysis revealed that all of the miRNAs identified in wild barley contained the highly conserved hairpin sequences found in barley cultivars. MiRNA target predictions showed that 12 out of 52 miRNA families were predicted to target transcription factors, including 8 highly conserved miRNA families in plants and 4 wheat-barley conserved miRNA families. In addition to transcription factors, other predicted target genes were involved in diverse physiological and metabolic processes and stress defense. Our study for the first time reported the large-scale investigation of small RNAs in wild barley, which will provide essential information for understanding the regulatory role of miRNAs in wild barley and also shed light on future practical utilization of miRNAs for barley improvement.