An increasing number of studies have reported that approximately 400 microRNAs (miRNAs), encoded by vertebrate viruses, regulate the expression of both host and viral genes. Many studies have used computational and/or experimental analyses to identify the target genes of miRNAs, thereby enabling us to understand miRNA functions. Here, we suggest that important aspects become apparent when we focus on conserved viral miRNAs, although these miRNA sequences generally show little similarity among viral species. Reliable viral miRNA-target gene pairs can be efficiently identified using evolutionary information. In this review, we summarize information on (i) the nucleotide sequence conservation among viral miRNAs and (ii) the RNAs targeted by viral miRNAs. Recent advances in these topics are discussed.