Abstract The monitoring of infectious diseases in wildlife is crucial for assessing animal health, pathogen range expansion, and the risk of spillover to naive species, but may be resource and labor intensive. Lymphoproliferative disease virus (LPDV) is an avian oncogenic retrovirus that was first identified in wild turkeys ( Meleagris gallopavo ) in 2009, though it historically caused mortality in domestic turkeys in Europe and Israel. Subsequent surveys detected a high prevalence and broad distribution throughout the eastern United States, warranting further research on LPDV in wild turkey populations. Current LPDV diagnostics require the collection of tissues, such as bone marrow from dead birds or blood during live capture. In our study, we assessed the sensitivity (true positive) and specificity (true negative) of cloacal swab samples as an alternative LPDV detection method. We compared results from cloacal swab samples with both postmortem detection from bone marrow and antemortem detection from blood, using a multi‐tube PCR approach with 3 replicates. Swab samples collected from live‐captured turkeys had a greater sensitivity (88%) than swabs collected from hunter‐harvested turkeys (31%), whereas specificity was similar for both collection approaches (live‐capture swabs = 75%, n = 85; hunter‐harvest swabs = 80%, n = 54). In live‐captured turkeys, the estimated LPDV prevalence using cloacal swab samples (73%) was not significantly different from the true prevalence determined using coupled blood samples (76%). However, in hunter‐harvested turkeys, the estimated prevalence using cloacal swab samples (28%) was different from the true prevalence estimated using coupled bone marrow samples (72%). In summary, cloacal swab samples can be used to reliably detect LPDV infection in live‐captured wild turkeys but should not be used for LPDV detection in hunter‐harvested wild turkeys.