A validated questionnaire assessing diabetic retinopathy (DR)-and diabetic macular edema (DME)-related knowledge (K) and attitudes (A) is lacking. We developed and validated the Diabetic Retinopathy Knowledge and Attitudes (DRKA) questionnaire and explored the association between K and A and the self-reported difficulty accessing DR-related information (hereafter referred to as Access). Methods: In this mixed-methods study, eight focus groups with 36 people with DR or DME (mean age, 60.1 ± 8.0 years; 53% male) were conducted to develop content (phase 1). In phase 2, we conducted 10 cognitive interviews to refine item phrasing. In phase 3, we administered 28-item K and nine-item A pilot questionnaires to 200 purposively recruited DR/DME patients (mean age, 59.0 ± 10.6 years; 59% male). The psychometric properties of DRKA were assessed using Rasch and classical methods. The association between K and A and DR-related Access was assessed using univariable linear regression of mean K/A scores against Access. Results: Following Rasch-guided amendments, the final 22-item K and nine-item A scales demonstrated adequate psychometric properties, although precision remained borderline. The scales displayed excellent discriminant validity, with K/A scores increasing as education level increased. Compared to those with low scores, those with high K/A scores were more likely to report better access to DR-related information, with K scores of 0.99 ± 0.86 for no difficulty; 0.79 ± 1.05 for a little difficulty; and 0.24 ± 0.85 for moderate or worse difficulty (P < 0.001). Conclusions: The psychometrically robust 31-item DRKA questionnaire can measure DR-and DME-related knowledge and attitudes. Translational Relevance: The DRKA questionnaire may be useful for interventions to improve DR-related knowledge and attitudes and, in turn, optimize health behaviors and health literacy.