Marek's disease (MD) is a major cause of mortality in backyard chickens. The diagnosis of MD is complex, however, and knowledge of Marek's disease virus (MDV) in spontaneous field cases such as in backyard chickens is largely unknown. In this study, 40 backyard chickens with a presumptive MD diagnosis based on histologic lymphoid infiltrations in peripheral nerves with and without lymphomas were investigated. Twenty-eight of the birds were submitted to the diagnostic laboratory for disease explorations, and 12 chickens were from a flock in which some members demonstrated anisocoria and pupil irregularities compatible with ocular MD. Histologic scores were established for brain, peripheral nerves, heart, lung, liver, kidney, and gonad sections, ranging from mild ( ) to severe ( ) lymphoid infiltrations. Twelve chickens had gross lymphomas, and all but two chickens had mild to severe peripheral nerve lymphoid infiltrates. There were no age or breed predispositions in the study group. Quantification of serotypes MDV-1, −2, and −3 performed with real-time PCR demonstrated high correlation (R² = 0.94) between fresh and fixed spleen specimens, as well as between histopathology scores and MDV-1 viral loads. MDV-2 DNA was detected in a portion of the chickens, likely consistent with naturally occurring virus, whereas the vaccine strain MDV-3 was rarely detected. Significant differences in MDV-1 viral loads between tumorous and nontumorous chickens were observed, in which a ratio of MDV-1 glycoprotein B/glyceraldehyde-3-phosphate dehydrogenase ≥ 0.5 was suggestive of gross tumors in this study. We propose that real-time PCR may be a good tool for MD diagnosis in backyard chickens.