Posterior tibial tendon dysfunction is one of the most common causes of acquired flat foot deformity in adults, and results in significant morbidity due to the pain and development of secondary osteoarthritis. The exact etiology of this condition is still unknown. Tibial tendons are predominately made up of fibrillar collagens and we hypothesis that their structural properties such as the collagen fiber density, orientation properties and cross-linking density essentially control the biomechanical properties of posterior tibial tendons. In this study, our aim is to visualize and quantitate fibrillar collagen distributions, their organization, crosslink densities and the fiber orientation (using Fourier analysis) in three distinct regions of posterior tibial tendon human samples namely proximal, middle and distal regions. The experiments conducted here are based on tendon specimens donated for research by persons that have their diseased tendon removed prior to surgery. Multiphoton microscopy provides a powerful imaging method for evaluation of remodeling of fibrillar collagen structures deep within tissues. Ultra-short IR laser pulses served as an excitation source to produce multiphoton excitation fluorescence (MPEF) from endogenously fluorescent macromolecular systems and to induce highly specific second harmonic generation (SHG) signals from fibrillar collagens. We systematically examined the nature of fibrillar collagen remodelling in relatively thick posterior tibial tendon tissues. Computed Orientation Index values obtained from Fourier analysis show statistically significant differences particularly between proximal and middle regions (p<0.0001). The crosslinking densities determined from the ratio of auto-fluorescence (MPEF) to BSHG again show differences particularly between proximal and middle regions (p<0.01). We have successfully demonstrated that the multiphoton and harmonic generation microscopy can be a powerful high resolution imaging method requiring minimal sample preparation that can provide structural information about spatially and spectrally resolved fibrillar collagens in three different posterior tibial tendon tendon regions.