The importance of the equine thoracolumbar vertebral column in orthopaedic disorders is well recognized and diagnostic imaging becomes more feasible, but little is known about variations in the anatomical configuration within breeds. In this descriptive post-mortem study, anatomical variations in three widely differing breeds: Warmblood horses, Shetland ponies and semi-feral Konik horses are described. The caudal cervical (C), thoracic (T), lumbar (L) and sacral (S) regions of the vertebral column of 30 Warmblood horses, 29 Shetland ponies and 18 Konik horses were examined using computed tomography and visualized by volume rendering. Homologous/morphologic variations in the caudal cervical area were frequently seen in Warmblood horses (43%), which was significantly more than in the other breeds (p < 0.001). The as standard described equine formula of 18 T, 6 L and 5 S vertebrae was seen in 78% of Konik horses, but only in 53% Warmblood horses and 38% Shetland ponies, which was significantly different (p < 0.05). Overall, Shetland ponies showed a higher tendency of thoracoization, lumbarization and more variations in the number of vertebrae and pairs of ribs. Ankylosed intertransverse joints (ITJs) between transverse processes of the lumbar vertebrae were most common between the second last and last lumbar vertebra and prevalence was significantly higher in Shetland ponies (61%), than in Warmblood horses (38%) and Konik horses (7%) (p < 0.0001). Cranial to the second last lumbar vertebra there were fewer ITJs ankylosed (14%) in Warmblood horses (p < 0.0095), and this decrease in number of ankylosed ITJs was different compared to the change in ankylosed ITJs in Shetland ponies (p < 0.005). ITJs occurred asymmetrically in 15% (12/77) of the cases. A limitation of the study was that clinical data of the horses were only incompletely available, precluding any conclusions about the potential clinical implications of anatomical variations. Knowledge of variation in osseous anatomy of the equine thoracolumbar vertebral column is important for the interpretation of diagnostic imaging. To assess the functional importance and clinical relevance of this variation, follow-up studies are necessary.