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Background Polymerase Chain Reaction (PCR) is a well-accepted adjunct in the management of infectious uveitis. In turn, few reports in the literature have evaluated how PCR then impacts patient care. This study aims to evaluate the impact of PCR sampling on diagnosis and treatment of infectious uveitidies at a large tertiary care facility. Main body This is a retrospective, observational study of patients with aqueous and vitreous PCR samples obtained from 2014 to 2019. The study was undertaken at a single institution. At least one follow up visit following results of PCR testing was required for inclusion. If a patient had multiple PCR samples taken, only the first sample was included. The patients were divided into three categories based on pre-sampling diagnosis. A chi-square test was used to analyze the data. 108 cases were available for analysis. PCR did not change diagnosis or management in any of the cases where pre-sampling diagnosis carried a high clinical suspicion for negative PCR. Overall, the results of PCR testing had a more significant impact on diagnosis in those cases where pre-sampling diagnosis was unknown versus those where it was confirmatory in nature, thus presumed to be related to an infectious entity tested by PCR (74% vs. 29%, p = 0.00006). The rate of treatment change based on PCR was similar between those cases where there was a high clinical suspicion for positive PCR and those where pre-sampling diagnosis was unknown (32% vs. 33%, p = 0.95). Further analyzing specimens separately depending on source of sample, this pattern persisted for aqueous samples, with PCR showing a more significant impact on diagnosis in those cases where the diagnosis was unknown versus those where sampling was confirmatory (86% vs. 31%, p = 0.00004). The rate of change in treatment between the two groups was similar (35% vs. 31%, p = 0.79). Vitreous samples followed a similar pattern with a higher rate of diagnosis change for those cases where pre-sampling diagnosis was unknown and a similar rate in treatment change between the two groups, however this did not reach statistical signifigance (44% vs. 25%, p = 0.28; 27% vs. 33%, p = 0.74). Conclusion There is no well-defined algorithm as to when to employ PCR testing in uveitis. As expected, in our experience, it has the largest impact on diagnosis when the diagnosis is unknown, however even when confirmatory in nature, it continues to impact patient management.