Cartilage canals (CCs) are microscopic structures involved in secondary ossification centers (SOCs) development. The features of CCs were investigated in the humeral and femoral proximal epiphyses of small-sized newborn dogs (from premature to 28 days after birth) with histochemical and immunohistochemical approaches. Masson’s Trichrome revealed a ring-shaped area around CCs, which changes in colour from green (immature collagen) to red (mature collagen) as ossification progresses; perichondrium staining always matched the ring colour. Safranin-O was always negative. Immunohistochemical analysis revealed immunopositivity for both collagen type I and V around the CCs; collagen type II was negative. CCs count showed a tendency to be higher in the humerus than in the femur. This work enlightened for the first time changes in composition of CCs surrounding matrix during SOCs development in dogs, paving the way to further investigations.