The aim of this study was to evaluate the effect of gestational weight gain (GWG) up to 23 weeks of gestation on the incidence of Gestational Diabetes Mellitus (GDM). A pregnant cohort of 5948 women in Northern Greece was recruited. Anthropometric features before and during pregnancy were recorded, the GWG by 23 weeks was calculated and a Generalized Linear Regression Model (GLM) with subgroup analyses based on weight status were computed. GDM was diagnosed in 5.5% of women. GLM results showed that GDM likelihood increased with maternal age (MA) and pre-pregnancy BMI (aOR: 1.08, 95%CI: [1.06, 1.11] and aOR: 1.09, 95%CI: [1.09, 1.11], respectively). Ιn the normal pre-pregnancy weight group, when the extra weight gain was >8 kgs, the odds of GDM increased (OR: 2.13, 95%CI: [0.98, 4.21], = 0.03). Women with pre-pregnancy level 2 clinical obesity (OB2 pre) (BMI > 35 and <40 kg/m) that shifted to OB3 category (BMI ≥ 40 kg/m) had an increased GDM likelihood (OR: 4.85, 95%CI: [1.50, 15.95]). Women of higher MA may require stricter monitoring for GDM from early pregnancy, while in obese women, recommended GWG may need to be re-evaluated, since refraining from any weight gain may have a preventive effect for GDM.