A reliable antemortem test is needed to understand the ecology of chronic wasting disease (CWD) in elk (Cervus elaphus nelsoni). We measured the ability of antemortem biopsy samples from the rectal mucosa to detect the abnormal prion protein associated with CWD (PrP^CWD), the relationship between test results from the obex and rectal biopsies at varying stages of CWD progression, and the prevalence of CWD in free-ranging elk from Rocky Mountain National Park, Colorado, USA. We sampled and placed radio collars on 136 adult female elk in the winter of 2007–08. Elk with biopsy samples found positive for PrP^CWD by immunohistochemistry (IHC) were euthanized and the obex and retropharyngeal lymph nodes were examined with IHC. We resampled, euthanized, and necropsied 20, 25, and 34 of the remaining study elk in each of the three following winters, respectively. Sensitivity of rectal biopsy samples increased in an asymptotic fashion with follicle count and was maximized at 85% (95% credible limits [CL]=60, 98) in the beginning of the study, when a greater proportion of elk were in a detectable stage of prion infection. However, maximum sensitivity was reduced to 72% (CL=46, 94) when we included resampled elk, which included recently infected elk that were initially negative using rectal biopsies and IHC. Test results were similar between rectal biopsies and the obex, but the earliest stages of prion infection were only detected by using retropharyngeal lymph nodes. Minimum CWD prevalence was estimated to be 9.9% (CL=5.7, 15.7) using rectal biopsies, but this rose to 12.9% (CL=8.0, 19.1) when we included four elk that were likely misdiagnosed at initial capture. Our results indicate rectal biopsies can provide a useful research tool for CWD in elk populations, but should be used with caution because they can miss individuals in early stages of infection and underestimate prevalence. Prevalence estimates from this population are the highest reported to date in elk and indicate that under appropriate conditions, CWD may be able to affect the dynamics of high-density elk populations.