The interfacial strength of secondary osteons from the diaphysis of the Thoroughbred equine third metacarpal was evaluated using the fiber pushout test. The pushout was performed on 300–500 μm sections of 4×4×15 mm bone blocks machined from four anatomic regions of the cortex. Pushout strength was evaluated from proximal to distal location within the diaphysis on four osteon types classified under polarized light on adjacent histologic sections from each block. The shear strength of the interfaces were estimated from shear lag theory. Differences were found in the interfacial strength of osteons based on appearance under polarized light with bright field having the highest interfacial strength (40.3 MPa). The lowest strength was found in the dark field osteons (22.8 MPa). The dorsal region had the highest shear strength and toughness compared to all other regions. The cement line and interlamellar interfaces are similar in strength, but exhibit regional dependence—specifically, the palmar region strength is less (17.5 MPa) than the osteon interlamellar interfaces (30.4 MPa) and osteon type dependent (alternating significantly weaker than other types). Histomorphometry revealed significant regional differences (p = 0.01) in osteon area fraction among the four osteon types as well as differences in the osteon diameter (p < 0.0001), with dorsal regions having larger osteons (170 μm) than the palmar region (151 μm). Fatigue life and fracture toughness of Haversian bone are reported in the literature to be regionally dependent and are known to be associated with osteon pullout—an osteon interfacial phenomenon. Therefore, the results presented in this study are important to further the understanding of the mechanisms of fragility and damage accumulation in cortical bone.