Purpose <p>To investigate distribution differences of parafoveal cone photoreceptors between dominant and non-dominant eyes using the adaptive optics scanning laser ophthalmoscope (AO-SLO) system.</p> Methods <p>This retrospective study included 156 eyes from 78 healthy subjects, all of whom had best-corrected visual acuity of 20/20 or better in both eyes and underwent ophthalmological examinations. The dominant eye was determined using the hole-in-the-card test. Retinal cone photoreceptor observations were performed at an eccentricity of 3.0° from the foveal center using the AO-SLO system. Quantitative distribution parameters, including cone density, spacing, regularity, and dispersion of both dominant and non-dominant eyes, were analyzed and compared.</p> Results <p>Dominant eyes exhibited higher cone photoreceptors density at a 3.0° horizontal eccentricity from the fovea [22,896 (20,954, 25,179) cells/mm²] than non-dominant eyes [22,621 (20,147, 24,253) cells/mm²] (<i>p</i> = 0.03). Correspondingly, the spacing of cone photoreceptors in dominant eyes [5.41 (5.09, 5.68) µm] was smaller than that in non-dominant eyes [5.47 (5.23, 5.72) µm] (<i>p</i> = 0.041). The interocular difference in cone regularity between dominant [96.53 (95.07, 97.46)%] and non-dominant [96.68 (95.49, 97.57)%] eyes was statistically significant (<i>p</i> = 0.04). However, there was no significant interocular difference in cone dispersion, which could reflect distribution uniformity (<i>p</i> = 0.795).</p> Conclusion <p>Our findings reveal that the dominant eye exhibits higher cone density in the parafoveal region with narrower spacing but a less regular arrangement than the non-dominant eye. These cytological features provide new evidence linking cone photoreceptor distribution to ocular dominance, which may account for the electrophysiological asymmetry observed between the two eyes.</p> Key messages <p><Emphasis Type="BoldItalic">What is known?</Emphasis></p> <p>• The adaptive optics scanning laser ophthalmoscope can correct aberrations caused by irregularities in the eye’s optics, allowing for the direct and noninvasive observation of retinal microstructures in vivo.</p> <p><Emphasis Type="BoldItalic">What is new?</Emphasis></p> <p>• Our study reveals that parafoveal cone photoreceptors in dominant eyes exhibit significantly higher density, narrower spacing, and a less regular arrangement compared to those in non-dominant eyes in vivo.</p> <p>• The cytological asymmetry between photoreceptor cells in dominant and non-dominant eyes may serve as a potential basis for the electrophysiological differences observed between the two.</p>