Several studies have indicated differences in bond strength of dental materials to crown and root dentin. To investigate the potential differences in matrix properties between these locations, we analyzed upper root and crown dentin in human third molars for ultimate tensile strength and collagen biochemistry. In both locations, tensile strength tested perpendicular to the direction of dentinal tubules (undemineralized crown = 140.4 ± 48.6/root = 95.9 ± 26.1; demineralized crown = 16.6 ± 6.3/root = 29.0 ± 12.4) was greater than that tested parallel to the tubular direction (undemineralized crown = 73.1 ± 21.2/root = 63.2 ± 22.6; demineralized crown = 9.0 ± 3.9/root = 16.2 ± 8.0). The demineralized specimens showed significantly greater tensile strength in root than in crown. Although the collagen content was comparable in both locations, two major collagen cross-links, dehydrodihydroxylysinonorleucine/its ketoamine and pyridinoline, were significantly higher in the root (by ~ 30 and ~ 55%, respectively) when compared with those in the crown. These results indicate that the profile of collagen cross-linking varies as a function of anatomical location in dentin and that the difference may partly explain the site-specific tensile strength.