Cattle disorders affecting the sphenoid sinus are underreported, likely due to difficulties in imaging and lacking topographic knowledge. This study aimed to provide a detailed description of the normal anatomical and morphometric features of the cattle sphenoid sinus. Twelve healthy adult Holstein cow heads were used, and the sinus was examined using CT, anatomical sectioning and 3D modelling. The sinus was bilaterally detected in all animals but exhibited structural asymmetry and significant interindividual differences in morphological characteristics. Three parts of the sinus were defined, namely the rostral, median and alar parts, which conform to the morphological structure of the sphenoid bone. The rostral part was bilateral in shape and located on the orbit wall of the presphenoid bone in all animals. The median part, which pneumatized the body of the sphenoid bone, was observed bilaterally in seven animals, while the alar part, which pneumatized the wing of the sphenoid bone, was formed bilaterally in four animals. The sinus volume and surface area were 11 ± 8 cm3 and 49 ± 30 cm2, respectively. These findings may aid in the diagnosis of cattle sphenoid sinus disorders and contribute to the knowledge of regional anatomy for radiologists and clinicians.