The utility of rectal lymphoid tissue sampling for the diagnosis of chronic wasting disease (CWD) infections in mule deer (Odocoileus hemionus) and white-tailed deer (Odocoileus virginianus) was evaluated. CWD-associated prion protein (PrP(CWD)) deposits were observed in the rectal mucosa from 19 orally inoculated mule deer by 381 days post-inoculation (p.i.); similarly, 45 out of 50 naturally infected mule deer had PrP(CWD) in their rectal mucosa. In orally inoculated white-tailed deer, the presence of glycine (G) or serine (S) at codon 96 of the native PrP (denoted 96GG, 96GS or 96SS) appeared to influence the temporal patterns of PrP(CWD) deposition: nine out of 11 infected 96GG individuals had PrP(CWD) in their rectal mucosa by 342 days p.i., whereas only three out of seven infected 96GS individuals had PrP(CWD) in their rectal mucosa by 381 days p.i. and none of three 96SS individuals had PrP(CWD) in their rectal mucosa by 751 days p.i. These findings support further evaluation of rectal mucosa sampling in CWD surveillance.