This study aimed to evaluate the physiological variations in the serum biochemical profile of neonate (3-5 days old) Senepol and crossbreed Holstein x Gir calves in Uberlândia, state of Minas Gerais, Brazil, and to compare values between breeds and sexes. In total, 168 healthy Senepol (n = 80, 28 females and 52 males) and crossbred Holstein x Gir (n = 88, 49 females and 39 males) calves were evaluated. From each animal, approximately 5 mL of blood was collected by jugular venipuncture into a sterile tube without anticoagulant to obtain serum. Serum concentrations of total protein, albumin, creatinine, urea, total cholesterol, triglycerides, calcium, and phosphorus; and the activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase, and gamma-glutamyl transferase (GGT) were determined on a automated multichannel analyzer. There were significant differences (p < 0.05) in serum concentrations for total protein, globulin, albumin/globulin ratio (A:G), creatinine, urea, phosphorus, calcium/phosphorus ratio (Ca:P), AST, and GGT between Senepol calves and crossbred Holstein x Gir calves, but no significant differences in serum concentrations were observed between male and female calves for either breed (p > 0.05). It was concluded that the serum biochemistry profile of newborn Senepol and crossbred Holstein x Gir calves was affected by differences in colostrum intake management and breed characteristics. Also, no differences in blood parameters were observed between male and female calves.