Background: The information about macular and retinal nerve fibre layer (RNFL) thickness in women with gestational diabetes mellitus (GDM) is scarce. GDM is a risk factor for development of type 2 diabetes mellitus. Purpose: The purpose of this study was to evaluate mean macular and RNFL thickness in pregnant women with GDM in a teaching institution in Malaysia. We also analyzed the association of age, HbA1c level, duration of GDM, type of treatment, family history, previous history of GDM and spherical equivalent with the macular and RNFL thickness. Patients and Methods: This was a prospective and cross-sectional study involving 78 pregnant women with GDM, 72 healthy pregnant and 70 healthy non-pregnant women. The study was conducted in Hospital Universiti Sains Malaysia from 2016 to 2018. Macular and RNFL thickness were measured during the third trimester using spectral-domain optical coherence tomography. Age, HbA1c level, duration of GDM, type of treatment, family history, previous history of GDM and spherical equivalent were analysed. Results: The mean macular thickness was 236.08 (16.44) µm, 237.26 (22.42) µm and 240.66 (20.95) µm for GDM, healthy pregnant, and healthy non-pregnant women. The mean RNFL thickness was 97.27 (9.14) µm, 99.83 (12.44) µm and 97.97 (10.07) µm for GDM, healthy pregnant, and healthy non-pregnant women. There was no significant difference in the mean macular and RNFL thickness in pregnant women with GDM when compared to the control groups (p>0.05). Age, HbA1c, duration of diabetes, treatment received, history of GDM and spherical equivalent did not show significant association with mean macular and retinal thickness (p>0.05). Conclusion: Pregnant women with GDM have similar thickness of the macular and RNFL with the healthy pregnant and healthy non-pregnant women. Age, HbA1c, duration of diabetes, treatment received, history of GDM and spherical equivalent showed no significant association with mean macular and retinal thickness in pregnant women with GDM.