Pregnancy in many mammals, including mares, is associated with physiological changes that are reflected in hematological and biochemical profiles. Understanding those physiological changes and differentiating them from pathological changes is imperative for providing care and medical therapy in pregnant mares. Our objective was to compare normal hematological and biochemical profiles in healthy non-pregnant as well as healthy pregnant mares during the 1st and 2nd trimesters of pregnancy. Blood was collected by jugular venipuncture into ethylenediaminetetraacetic acid and serum tubes. Whole blood was analyzed using an ADVIA 120 hematologic analyzer and serum was analyzed using a Beckman Coulter AU5400. Statistical differences were detected using analysis of variance (ANOVA) and independent Student’s t-test; P < 0.05 was considered significant. Results demonstrated higher red blood cell counts and hemoglobin concentrations and lower eosinophil counts (P < 0.001) in non-pregnant mares (n = 32) compared with pregnant mares at both 1st (n = 25) and 2nd (n = 17) trimesters. Biochemical analysis showed a significant decrease (P < 0.001) in albumin and blood urea nitrogen (P < 0.001) in the 2nd trimester and higher potassium levels (P = 0.03) in pregnant mares. Understanding such physiological changes is imperative to providing optimum care and medical treatment in mares. These data will assist clinicians to better evaluate and treat pregnant mares.