MicroRNAs (miRNAs) are a class of non-coding RNAs that play critical roles in post-transcriptional regulation. Their target genes are involved in a variety of biological processes such as development, metabolism, and stress response. Panicum miliaceum L. (Panicum) is an important grain crop, but, until now, no miRNAs have been identified in this plant. Using a homology search based on expressed sequence tag (EST) analysis and miRNA precursor secondary structure, a total of 43 new miRNAs were identified. The miRNAs were found to be unevenly distributed among 11 miRNA families. Target analysis using the plant small RNA target analysis server psRNATarget showed that the newly identified miRNAs can potentially regulate 68 target genes. Ten of the 11 miRNA families were annotated as involved in RNA regulation, suggesting they may play an essential role in post-transcriptional regulation in Panicum. Selected miRNAs representing eight of the families were verified by northern blotting, indicating that the prediction method that we used to identify the miRNAs was effective.