To identify the prominent factors that lead to misdiagnosis of retinopathy of prematurity (ROP) by ophthalmologists-in-training in the United States and Canada. This prospective cohort study included 32 ophthalmologists-in-training at six ophthalmology training programs in the United States and Canada. Twenty web-based cases of ROP using wide-field retinal images were presented, and ophthalmologists-in-training were asked to diagnose plus disease, zone, stage, and category for each eye. Responses were compared to a consensus reference standard diagnosis for accuracy, which was established by combining the clinical diagnosis and the image-based diagnosis by multiple experts. The types of diagnostic errors that occurred were analyzed with descriptive and chi-squared analysis. Main outcome measures were frequency of types (category, zone, stage, plus disease) of diagnostic errors; association of errors in zone, stage, and plus disease diagnosis with incorrectly identified category; and performance of ophthalmologists-in-training across postgraduate years. Category of ROP was misdiagnosed at a rate of 48%. Errors in classification of plus disease were most commonly associated with misdiagnosis of treatment-requiring (plus error rate = 16% when treatment-requiring was correctly diagnosed vs 81% when underdiagnosed as type 2 or pre-plus; mean difference: 64.3; 95% CI: 51.9 to 76.7; P < .001) and type 2 or pre-plus (plus error rate = 35% when type 2 or pre-plus was correctly diagnosed vs 76% when overdiagnosed as treatment-requiring; mean difference: 41.0; 95% CI: 28.4 to 53.5; P < .001) disease. The diagnostic error rate of postgraduate year (PGY)-2 trainees was significantly higher than PGY-3 trainees (PGY-2 category error rate = 61% vs PGY-3 = 35%; mean difference, 25.4; 95% CI: 17.7 to 33.0; P < .001). Ophthalmologists-in-training in the United States and Canada misdiagnosed ROP nearly half of the time, with incorrect identification of plus disease as a leading cause. Integration of structured learning for ROP in residency education may improve diagnostic competency. [J Pediatr Ophthalmol Strabismus. 2023;60(5):337-343.].