Diabetic macular ischemia (DMI) is a phenotype of diabetic retinopathy (DR) associated with chronic hypoxia of retinal tissue. The goal of this prospective observational study was to report evidence of photoreceptor abnormalities using adaptive optics scanning laser ophthalmoscopy (AOSLO) in eyes with DR in the setting of deep capillary plexus (DCP) non-perfusion. Eleven eyes from 11 patients (6 women, age 31–68), diagnosed with DR without macular edema, underwent optical coherence tomography angiography (OCTA) and AOSLO imaging. One patient without OCTA imaging underwent fluorescein angiography to characterize the enlargement of the foveal avascular zone. The parameters studied included photoreceptor heterogeneity packing index (HPi) on AOSLO, as well as DCP non-perfusion and vessel density on OCTA. Using AOSLO, OCTA and spectral domain (SD)-OCT, we observed that photoreceptor abnormalities on AOSLO and SD-OCT were found in eyes with non-perfusion of the DCP on OCTA. All eight eyes with DCP non-flow on OCTA showed photoreceptor abnormalities on AOSLO. Six of the eight eyes also had outer retinal abnormalities on SD-OCT. Three eyes with DR and robust capillary perfusion of the DCP had normal photoreceptors on SD-OCT and AOSLO. Compared to eyes with DR without DCP non-flow, the eight eyes with DCP non-flow had significantly lower HPi (P = 0.013) and parafoveal DCP vessel density (P = 0.016). We found a significant correlation between cone HPi and parafoveal DCP vessel density (r = 0.681, P = 0.030). Using a novel approach with AOSLO and OCTA, this study shows an association between capillary non-perfusion of the DCP and abnormalities in the photoreceptor layer in eyes with DR. This observation is important in confirming the significant contribution of the DCP to oxygen requirements of photoreceptors in DMI, while highlighting the ability of AOSLO to detect subtle photoreceptor changes not always visible on SD-OCT.