The gene MTNR1B encodes a receptor for melatonin. Melatonin receptors are expressed in human β-cells, which implies that genetic variants might affect glucose tolerance. Meta-analysis confirmed that the rs10830963 shows the most robust association. The aim of the study was to assess the rs10830963 in Czech GDM patients and controls and to study relations between the SNP and biochemical as well as anthropometric characteristics. Our cohort consisted of 880 women; 458 were diagnosed with GDM, and 422 were normoglycemic controls without history of GDM. Despite similar BMI, the GDM group showed higher WHR, waist circumference, abdominal circumference, and total body fat content. The risk allele G was more frequent in the GDM group (38.3 versus 29.4% in controls, OR 1.49 CI95% [1.22; 1.82]; P OR = 0.0001). In spite of higher frequency, the G allele in the GDM group was not associated with any markers of glucose metabolism. In contrast, controls showed significant association of the allele G with FPG and with postchallenge glycemia during the oGTT. Frequency analysis indicates that rs10830963 is involved in gestational diabetes in Czech women. However, the association of the SNP with glucose metabolism, which is obvious in controls, is covert in women who have experienced GDM.