Objective: This study aimed to review the authors' results using polymerase chain reaction (PCR)-based assays for the diagnosis of viral retinitis. Design: The study design was a retrospective case series. Participants: Thirty-seven patients (38 eyes) with active retinitis from whom vitreous biopsy specimens were received in the authors' laboratory for diagnostic evaluation. Intervention: Vitreous biopsy specimens were evaluated with previously described PCR-based assays for cytomegalovirus (CMV), varicella zoster virus (VZV), and herpes simplex virus (HSV) DNA; clinical histories were reviewed. Main Outcome Measures: Laboratory findings and clinical course were measured. Results: The results of the authors' assays were consistent with the long-term clinical course of each patient. Cytomegalovirus, VZV, or HSV DNA was detected in the vitreous from 24 patients. Cytomegalovirus DNA was detected in vitreous biopsy specimens from 10 patients (11 eyes). Nine patients (ten eyes) with acquired immune deficiency syndrome ultimately were diagnosed with CMV retinitis as they were followed clinically over time. Varicella zoster virus DNA was detected in vitreous biopsy specimens from eight patients; seven adult patients were ultimately diagnosed with acute retinal necrosis or progressive outer retinal necrosis. Herpes simplex virus DNA was detected in vitreous biopsy specimens from six patients; five patients had previous or subsequent herpes encephalitis. No viral DNA was detected in the vitreous from 13 patients; all were ultimately diagnosed with toxoplasmosis, syphilis, Behcet disease, fungal endophthalmitis, or idiopathic inflammation. Conclusions: These data further support the use of PCR-based assays of vitreous specimens in the diagnostic evaluation of patients with infectious retinitis.