This study describes the first recognized clinical case of lymphoproliferative disease virus (LPDV) in Canada and extends the range of LPDV in Canada through its detection in Manitoba and Quebec. We assessed the prevalence of LPDV in eastern wild turkeys (Meleagris gallopavo silvestris) with the use of whole, clotted blood from live birds in Manitoba (n = 65) and tissue samples collected postmortem in Quebec (n = 4). We tested for LPDV proviral DNA through PCR amplification and sequencing of a portion of the gag (p31) gene. Samples were also tested for reticuloendotheliosis virus (REV) by PCR. Twenty-four birds (34.8%) were positive for LPDV, including all diagnostic cases. One bird (1.4%) from Quebec had gross and microscopic lesions consistent with LPDV. Two turkeys (2.9%) were REV positive, one (1.4%) of which was co-infected with LPDV. Phylogenetic analysis of LPDV strains from Quebec and Manitoba grouped with previously sequenced samples from Ontario and publicly available sequences from a North American lineage. This study contributes valuable information toward ongoing surveillance and monitoring of LPDV in North America.