To assess polymerase chain reaction (PCR) analysis of intraocular fluid as a test for infectious uveitis of the posterior segment in a representative patient population. Retrospective, interventional case series. One hundred and thirty-three patients with possible infectious chorioretinitis underwent PCR testing of aqueous or vitreous in a university setting. Baseline characteristics predictive of test positivity were identified. Positive and negative predictive values were calculated. Four hundred and thirty-three PCR tests of 105 aqueous and 38 vitreous specimens (mean, 3.3 tests per patient) identified 77 of the 95 patients with a final clinical diagnosis of infectious uveitis (81%). Herpes simplex virus, varicella zoster virus, and cytomegalovirus PCR analysis were performed in almost all cases, with fewer tests for toxoplasmosis or Epstein-Barr virus. Clinical features associated with positive PCR results were retinal vascular inflammation (P < .001), optic nerve involvement (P = .008), immunocompromised state (P = .039), and extensive retinitis (P = .002). Cases sampled within one week of presentation were more likely to have positive PCR results than those sampled later (P = .071). The predictive value of positive and negative tests was 98.7% and 67.9%, respectively, in this patient group. Alteration in treatment based on PCR and syphilis serologic results led to resolution in 25 of 26 patients after treatment was changed. PCR testing is a useful adjunct in the diagnosis of infectious causes of posterior uveitis. Cases with vascular or optic nerve inflammation, extensive retinitis, or immunocompromise are more likely to have positive PCR results and may benefit from PCR testing of aqueous humor.