microRNAs (miRNAs) are post-transcriptional regulators of gene expression that play a role in viral infection. We have developed a method to identify viral-encoded miRNAs from viruses in which abundant amounts of infected material is limiting. We show that Merkel Cell Polyomavirus (MCV), a recently identified human virus associated with cancer, encodes a miRNA. This miRNA is expressed from the late strand, lies antisense to the early transcripts and negatively regulates expression of chimeric reporters containing a portion of the early transcripts. Interestingly, different viral isolates have sequence polymorphisms in the pre-miRNA region that result in amino acids substitutions but fully preserve the processing and activity of the miRNAs.