In an effort to systematically describe the neurochemical anatomy of the bovine anterior hypothalamus, we used a series of immunocytochemical markers such as Acetylcholine Esterase (AChE), Arginine-Vasopressin (AVP), Calbindin (Calb), Galanin (Gal), Neuropeptide-Y (NPY), Oxytocin (OXT), Somatostatin (SST) and Vasoactive Intestinal Peptide (VIP). We also investigated the potential sex difference present in the suprachiasmatic nucleus (SCN) and the vasopressin-oxytocin containing nucleus (VON) of 6 males and 6 female Bos taurus. Our study revealed that the cytochemical structure of the cattle anterior hypothalamus follows the blueprint of other mammals. The VON, which was never described before in cattle, showed a sex difference with a 33.7% smaller volume and 23.2% fewer magnocellular neurons (approximately 20-30 μm) in the male. The SCN also did show a sex difference in VIP neurons and volume with a 36.1% larger female nucleus with 28.1% more cells. Additionally, we included 5 heifers with freemartin syndrome as a new animal model relevant for sexual differentiation in the brain. This is to our knowledge the first freemartin study in relation to the brain. Surprisingly, the SCN of freemartin heifers was 32.5% larger than its control male and female counterparts with 29% more VIP cells. Conversely, the freemartin VON had an intermediary size between male and female. To analyze our data, a classical statistical analysis and a novel multivariate and multi-aspect approach were applied. These findings shed new light on sexual dimorphism in the bovine brain and present this species with freemartins as a valuable animal model in neuroscience. This article is protected by copyright. All rights reserved.