Purpose This study aims to determine the effect of serum free triiodothyronine (fT3), free thyroxine (fT4), thyroid-stimulating hormone (TSH) and fT3/fT4 ratio on the gestational diabetes. Methods This multicenter study was conducted by analyzing retrospectively 495 pregnant women who admitted to the Tepecik training and Research Hospital, and Kocaeli Derince training and Research Hospital between January 2017 and December 2019. Seventy-eight pregnant women diagnosed with Gestational Diabetes Mellitus (GDM) and 82 pregnant women without GDM diagnosis were included in the study. The relationship between thyroid markers (TSH, fT3, fT4 and fT3/fT4) and gestational diabetes was determined using logistic regression analysis. Results Advanced age, low fT4 and high TSH levels were higher in GDM group, p < .05. Results of the logistic regression analysis revealed that the gestational week (OR 20.291, 95% CI 3.132-131-449, p = .002], fasting plasma glucose (OR 48,551, 95% CI 9.223-255.582, p < .001), HbA1c (OR 13,671, 95%CI 2.977-62.781, p =.001) and fT3/fT4 (OR 29.934, 95% CI 3.558-251836, p = .002) were the risk factors of GDM. Discussion Advanced gestational week, fasting blood glucose, HbA1c and fT3/fT4 ratio are among the risk factors for the development of the gestational diabetes.