Purpose: The Pediatric Vision Scanner (PVS) is a binocular retinal birefringence scanner designed to identify young children with amblyopia and strabismus, two common causes of preventable vision loss. While the PVS has been shown to be highly sensitive and specific in an enriched population, validation is lacking in a general pediatric setting. We conducted a prospective clinical trial to assess the performance of the PVS in a non-enriched population. Methods: We recruited 300 children ages 2 to 5 years with no pre-existing eye conditions from Kaiser Permanente Southern California’s Orange County service area. Each participant attended a single study visit at one of two ophthalmology clinics. Trained research staff screened participants with the PVS which displayed a pass/refer result. A pediatric ophthalmologist then performed a gold standard eye exam while masked to the PVS result. To compare the PVS results to the gold standard eye exam, we performed a validation characteristics analysis with a 95% confidence interval (CI). We also measured child cooperation during the PVS screening (“excellent” or “fair”) and acquisition time of the PVS (time between when the research staff picks up the PVS to when the exam is complete). Results: Based on the gold standard eye exam, 6 children (2%) had amblyopia. Of these, there were 2 children (0.67%) who also had strabismus. The PVS referred all 6 of these patients, yielding a sensitivity rate of 100% (95% CI: [54%, 100%]). The PVS referred 45 additional children (15%) who had normal ophthalmic findings, yielding a specificity rate of 85% (95% CI: [80%, 89%]). When stratified by cooperation, specificity increased to 91% (95% CI: [86%, 95%]) among the 178 children (59.3%) with “excellent” cooperation. Specificity did not change when stratified by child’s age. The overall positive predictive value was 12% (95% CI: [4%, 24%]), and the negative predictive value was 100% (95% CI: [99%, 100%]). The median acquisition time for the PVS was 28 seconds. Conclusion: Given its short acquisition time, PVS screening can be implemented in a pediatric clinic with minimal impact on workflow. This would allow children with amblyopia and/or strabismus to be referred to an eye care specialist as early as two years old. The 15% false positive rate is similar to what has been reported in previous studies. These results also suggest that device validity increases with cooperation (i.e., when the child keeps his/her head still for the duration of the exam) regardless of child’s age, which may be useful knowledge for clinical implementation. The negative predictive value results suggest that a “pass” result is highly reliable. These findings are the largest PVS results available in a non-enriched population.