© 2022 Elsevier B.V.Background To investigate whether single retinal layer, choroidal and peripapillary retinal fiber layer thicknesses were affected in patients under hydroxychloroquine treatment without retinal toxicity Methods Forty-nine patients under hydroxychloroquine treatment (HCQ group) and 39 healthy volunteers, a total of 88 individuals, were included in the study. Patients with HCQ treatment were further divided into two groups as less than 5 years of HCQ use (group 1, 29 patients) and 5 years or more of HCQ use (group 2, 20 patients). All participants underwent full ophthalmologic examination and optical coherence tomography (OCT) imaging as OCT is a recommended tool for screening toxicity by the American Association of Ophthalmology. The automatic segmentation of retinal layers included retinal nerve fiber layer (RNFL), ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL), outer nuclear layer (ONL), retina pigment epithelium (RPE), inner retinal layers (IRL), outer retinal layers (ORL) and total retina thickness (TRT). Choroidal thickness (ChT), choroidal vascularity index and peripapillary RNFL were also measured. Results Both inferior and nasal parafoveal RNFL, temporal parafoveal GCL, and IPL layer thicknesses were decreased, while temporal parafoveal RPE layer thicknesses were increased in hydroxychloroquine users. Subfoveal ChTs were significantly reduced in both groups 1 and 2 compared to the control group(p = 0.042 and p = 0.009, respectively) and temporal ChT thicknesses were statistically decreased in group 2 compared to the control group(p = 0.018), but not for nasal ChT. Conclusions Hydroxychloroquine use may cause parafoveal retinal layer thickness alterations and also subfoveal and parafoveal ChT alterations even without evident retinal toxicity in OCT.