AbstractMicroRNAs (miRNAs), a class of short (20–24 nt) non‐coding RNAs that direct post‐transcriptional repression of messenger RNAs, increasingly have been shown to play a key role in regulating cellular physiology. We investigated the prevalence of miRNAs in Chinese hamster ovary (CHO) cells by high‐throughput sequencing. Six cDNA libraries of small RNAs from four CHO cell lines were constructed and sequenced by Illumina sequencing. Three hundred fifty distinct miRNA and miRNA* sequences were identified through homology with other species, including mouse, rat, and human. While the majority of the identified miRNAs appear to be expressed ubiquitously, many miRNAs were found to have a wide range of expression levels between cell lines. The identification of these miRNAs will facilitate investigations of their contribution to the hyperproductivity trait. Biotechnol. Bioeng. 2011;108: 475–480. © 2010 Wiley Periodicals, Inc.