Multi-modal multiphoton microscopy was used to investigate tissue microstructure in the zone of calcified cartilage, focussing on the collagen fibre organisation at the tidemark and cement line. Thick, unstained and unfixed sagittal sections were prepared from the equine metacarpophalangeal joint. Second harmonic generation (SHG) provided contrast for collagen, two-photon fluorescence (TPF) for endogenous fluorophores, and coherent anti-Stokes Raman scattering (CARS) allowed the cells to be visualised. The structure of radial and calcified cartilage was found to vary with location across the joint, with the palma regions showing a more ordered parallel arrangement of collagen fibres than the cortical ridge and dorsal regions. These patterns may be associated with regional variations in joint loading. In addition, the cell lacunae had a greater diameter in the dorsal region than in the palmar region. At the cement line some collagen fibres were observed crossing between the calcified cartilage and the subchondral bone. At the tidemark the fibres were parallel and continuous between the radial and calcified cartilage. Beneath early superficial lesions the structure of the tidemark and calcified cartilage was disrupted with discontinuities and gaps in the fibrillar organisation. Cartilage microstructure varies in the deep zones between regions of different loading. The variations in collagen structure observed may be significant to the local mechanical properties of the cartilage and therefore may be important to its mechanical interactions with the subchondral bone. The calcified cartilage is altered even below early superficial lesions and therefore is important in the understanding of the aetiology of osteoarthritis.