Cotton-wool spots (CWSs) are common retinal manifestations of many diseases including diabetes mellitus, systemic hypertension, and acquired immunodeficiency syndrome. Clinically they appear as whitish, fluffy patches on the retina and eventually fade with time. In this study, spectral domain optical coherence tomography (SD-OCT) with mapping was used to demonstrate in vivo the characteristics of an isolated CWS in a 59-year-old patient as well as its appearance immediately after ophthalmoscopic resolution. Presented here is the work-up and management of this clinical problem for the ophthalmologist. The authors propose that SD-OCT could be a valuable research tool in characterizing and following the dynamic CWS changes at individual retinal layer level, with potential clinical applications as a screening or diagnostic tool in CWS-related diseases.