Chronic wasting disease (CWD) is an emerging infectious prion disorder that is spreading rapidly in wild populations of cervids in North America. The risk of zoonotic transmission of CWD is as yet unclear but a high priority must be to minimize further spread of the disease. No simple diagnostic tests are available to detect CWD quickly or in live animals; therefore, easily accessible biomarkers may be useful in identifying infected animals. MicroRNAs (miRNAs) are a class of small, non-coding RNA molecules that circulate in blood and are promising biomarkers for several infectious diseases. In this study we used next-generation sequencing to characterize the serum miRNA profiles of 35 naturally infected elk that tested positive for CWD in addition to 35 elk that tested negative for CWD. A total of 21 miRNAs that are highly conserved amongst mammals were altered in abundance in sera, irrespective of hemolysis in the samples. A number of these miRNAs have previously been associated with prion diseases. Receiver operating characteristic (ROC) curve analysis was performed to evaluate the discriminative potential of these miRNAs as biomarkers for the diagnosis of CWD. We also determined that a subgroup of 6 of these miRNAs were consistently altered in abundance in serum from hamsters experimentally infected with scrapie. This suggests that common miRNA candidate biomarkers could be selected for prion diseases in multiple species. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses pointed to a strong correlation for 3 of these miRNAs, miR-148a-3p, miR-186-5p, miR-30e-3p, with prion disease.