Gestational Diabetes Mellitus is known as carbohydrate intolerance first detected during pregnancy. Pregnancy is periods of intense hormonal changes. The aim of the present study was to investigate a possible relation between the changes in serum hormones such as Luteinizing hormone (LH) , follicle stimulating hormone(FSH), Progesterone, and Prolactin with gestational diabetes mellitus. Thirty patients with gestational diabetes mellitus aged (22 -40) year attending the national center for treatment and research of diabetes/ AL-Mustansiriya University in Baghdad and 29 controls aged (20-39) year were participated. Hormonal tests including, FSH, LH, Progesterone, and Prolactin were detected by using Enzyme Linked Fluorescent Assay (ELFA) kits. The demographic characteristics of gestational diabetes mellitus indicated  that the most commonly affected age at (20-29) year (50%) ,60% of patients had Body Mass Index(BMI)  at ≥ 30kg/m2+, 76.6% of patients at  first trimester of pregnancy, 23.3% had previous abortion,60% at the first pregnancy ,and 46.6%  of patients  had urine protein with one plus. A highly significant increase (p≤0.001) in Fasting serum glucose(FSG), LH, FSH, progesterone, and prolactin were observed in sera of gestational diabetes mellitus patients in comparison to that of control pregnancy group. A non-significant correlation of FSG with age, BMI, LH, FSH, and progesterone were demonstrated. While a significant positive correlation of FBS with prolactin was found. It is conclude that higher prolactin level in pregnancy possibly played a role in gestational diabetes mellitus partly by impairing the functions of insulin, and result in hyperglycemia. Keyword: Gestational diabetes, FSH, LH, Progesterone, Prolactin.