As the longissimus dorsi muscle is the largest muscle in the equine back, it has great influence on the stability of the spine and facilitates proper locomotion. The longissimus muscle provides support to the saddle and rider and thereby influences performance in the horse. Muscular dysfunction has been associated with back disorders and decline of performance. In general, muscle function is determined by its specific intramuscular architecture. However, only limited three-dimensional metrical data are available for the inner organisation of the equine longissimus dorsi muscle. Therefore, we aimed at investigating the inner architecure of the equine longissimus. The thoracic and lumbar longissimus muscles of five formalin-fixed cadaveric horse backs of different ages and body types were dissected layerwise from cranial to caudal. Three-dimensional coordinates along individual muscle fibre bundles were recorded using a digitisation tool (MicroScribe®), to capture their origin, insertion and general orientation. Together with skeletal data from computed tomography (CT) scans, 3D models were created using imaging software (Amira). For further analysis, the muscle was divided into functional compartments during preparation and morphometric parameters, such as the muscle fascicle length, pennation angles to the sagittal and horizontal planes, muscle volume and the physiological cross-sectional area (PCSA), were determined. Fascicle length showed the highest values in the thoracic region and decreased from cranial to caudal, with the cranial lumbar compartment showing about 75% of cranial fascicle length, while in most caudal compartments, fascicle length was less than 50% of the fascicle length in thoracic compartments. The pennation angles to the horizontal plane show that there are differences between compartments. In most cranial compartments, fascicles almost run parallel to the horizontal plane (mean angle 0°), while in the caudal compartment, the angles increase up to a mean angle of 38°. Pennation angles to the sagittal plane varied not only between compartments but also within compartments. While in the thoracic compartments, the fascicles run nearly parallel to the spine, in the caudal compartments, the mean angles range from 0-22°. The muscle volume ranged from 1350 cm3 to 4700 cm3 depending on body size. The PCSA ranged from 219 cm2 to 700 cm2 depending on the muscle volume and mean fascicle length. In addition to predictable individual differences in size parameters, there are obvious systemic differences within the muscle architecture along the longissimus muscle which may affect its contraction behaviour. The obtained muscle data lay the anatomical basis for a specific biomechanical model of the longissimus muscle, to simulate muscle function under varying conditions and in comparison to other species.