The aim of this study was to quantify the distribution of aligned and non-aligned collagen in cross-sections of the superficial digital flexor tendon (SDFT) and deep digital flexor tendon (DDFT) in different levels of the distal forelimb of equines diagnosed with NS (Navicular Syndrome). Sixty equine forelimbs were collected. Was compared two groups (NA, Not affected vs. NS-group) by t-Student. Diagnosis of NS was based on clinical and lameness examination, diagnostic analgesia and radiological findings. The proportion of aligned and non-aligned collagens at 2 levels for the SDFT and 3 levels for the DDFT were measured by histochemical stains. The amount of aligned and non-aligned collagen in tendons were calculated using Colour-Based Segmentation function. Regarding collagen, there were significant differences in the amount of aligned collagen (NA: 21.2 ± 1.31; NS-group: 12.2 ± 4.67; p = 0.0026) and non-aligned collagen (NA: 21.8 ± 2.22; NS: 25.1 ± 1.73; p = 0.0241) at the DDFT insertion in the distal phalanx. We concluded that the flexor tendons of the forelimb in equines with NS have different proportions of collagen than those that do not present the diagnosis, indicated by histologically visible increased proportions of non-aligned collagen and decreased of aligned collagen in the extracellular matrix.