Light intensity varies 1 million-fold between night and day, driving the evolution of eye morphology and retinal physiology. Despite extensive research across taxa showing anatomical adaptations to light niches, surprisingly few empirical studies have quantified the relationship between such traits and the physiological sensitivity to light. In this study, we employ a comparative approach in frogs to determine the physiological sensitivity of eyes in two nocturnal (Rana pipiens, Hyla cinerea) and two diurnal species (Oophaga pumilio, Mantella viridis), examining whether differences in retinal thresholds can be explained by ocular and cellular anatomy. Scotopic electroretinogram (ERG) analysis of relative b-wave amplitude reveals 10- to 100-fold greater light sensitivity in nocturnal compared to diurnal frogs. Ocular and cellular optics (aperture, focal length, and rod outer segment dimensions) were assessed via the Land equation to quantify differences in optical sensitivity. Variance in retinal thresholds was overwhelmingly explained by Land equation solutions, which describe the optical sensitivity of single rods. Thus, at the b-wave, stimulus-response thresholds may be unaffected by photoreceptor convergence (which create larger, combined collecting areas). Follow-up experiments were conducted using photopic ERGs, which reflect cone vision. Under these conditions, the relative difference in thresholds was reversed, such that diurnal species were more sensitive than nocturnal species. Thus, photopic data suggest that rod-specific adaptations, not ocular anatomy (e.g., aperture and focal distance), drive scotopic thresholds differences. To the best of our knowledge, these data provide the first quantified relationship between optical and physiological sensitivity in vertebrates active in different light regimes.