There is no early, first-trimester risk estimation available to predict later (gestational week 24-28) gestational diabetes mellitus (GDM); however, it would be beneficial to start an early treatment to prevent the development of complications. We aimed to identify early, first-trimester prediction markers for GDM. The present case-control study is based on the study cohort of a Hungarian biobank containing biological samples and follow-up data from 2545 pregnant women. Oxidative-nitrative stress-related parameters, steroid hormone, and metabolite levels were measured in the serum/plasma samples collected at the end of the first trimester from 55 randomly selected control and 55 women who developed GDM later. Pregnant women who developed GDM later during the pregnancy were older and had higher body mass index. The following parameters showed higher concentration in their serum/plasma samples: fructosamine, total antioxidant capacity, testosterone, cortisone, 21-deoxycortisol; soluble urokinase plasminogen activator receptor, dehydroepiandrosterone sulfate, dihydrotestosterone, cortisol, and 11-deoxycorticosterone levels were lower. Analyzing these variables using a forward stepwise multivariate logistic regression model, we established a GDM prediction model with a specificity of 96.6% and sensitivity of 97.5% (included variables: fructosamine, cortisol, cortisone, 11-deoxycorticosterone, SuPAR). Based on these measurements, we accurately predict the development of later-onset GDM (24th-28th weeks of pregnancy). Early risk estimation provides the opportunity for targeted prevention and the timely treatment of GDM. Prevention and slowing the progression of GDM result in a lower lifelong metabolic risk for both mother and offspring.