MicroRNAs (miRNAs) have recently entered Chinese hamster ovary (CHO) cell culture technology, due to their severe impact on the regulation of cellular phenotypes. Applications of miRNAs that are envisioned range from biomarkers of favorable phenotypes to cell engineering targets. These applications, however, require a profound knowledge of miRNA sequences and their genomic organization, which exceeds the currently available information of ~400 conserved mature CHO miRNA sequences. Based on these recently published sequences and two independent CHO-K1 genome assemblies, this publication describes the computational identification of CHO miRNA genomic loci. Using BLAST alignment, 415 previously reported CHO miRNAs were mapped to the reference genomes, and subsequently assigned to a distinct genomic miRNA locus. Sequences of the respective precursor-miRNAs were extracted from both reference genomes, folded in silico to verify correct structures and cross-compared. In the end, 212 genomic loci and pre-miRNA sequences representing 319 expressed mature miRNAs (approximately 50% of miRNAs represented matching pairs of 5' and 3' miRNAs) were submitted to the miRBase miRNA repository. As a proof-of-principle for the usability of the published genomic loci, four likely polycistronic miRNA cluster were chosen for PCR amplification using CHO-K1 and DHFR (-) genomic DNA. Overall, these data on the genomic context of miRNA expression in CHO will simplify the development of tools employing stable overexpression or deletion of miRNAs, allow the identification of miRNA promoters and improve detection methods such as microarrays.