MicroRNAs (miRNAs) are a newly identified class of non-protein-coding small RNAs, which play important roles in multiple biological and metabolic processes at the post-transcriptional level by directly cleaving targeted mRNAs or inhibiting translation. The lengths of viral miRNA precursors vary from 60 to 119 with an average of 79 nucleotides, which was smaller than observed for plant or animal miRNAs. Viral miRNAs are less conserved than plant and animal miRNAs, suggesting that viral miRNAs may evolve rapidly. Uracil nucleotide was highly dominant in the first position of 5′ mature miRNAs, Viral miRNAs had high minimal folding free energy index (MFEI, 0.9 ± 0.1). Based on these features and the well-known characteristics of miRNAs, 20 new potential miRNAs were identified in viruses by using expressed sequence tag (EST) analysis and genomic sequence survey (GSS) analysis. A better understanding of viral miRNA functions will be useful to design new approaches for treating viruses, especially those viruses that can induce human, animal, and plant diseases.