Collagen is the most abundant protein of the organic matrix in mineralizing tissues. One of its most critical properties is its cross‐linking pattern. The intermolecular cross‐linking provides the fibrillar matrices with mechanical properties such as tensile strength and viscoelasticity. In this study, Fourier transform infrared (FTIR) spectroscopy and FTIR imaging (FTIRI) analyses were performed in a series of biochemically characterized samples including purified collagen cross‐linked peptides, demineralized bovine bone collagen from animals of different ages, collagen from vitamin B₆‐deficient chick homogenized bone and their age‐ and sex‐matched controls, and histologically stained thin sections from normal human iliac crest biopsy specimens. One region of the FTIR spectrum of particular interest (the amide I spectral region) was resolved into its underlying components. Of these components, the relative percent area ratio of two subbands at ∼1660 cm⁻¹ and ∼1690 cm⁻¹ was related to collagen cross‐links that are abundant in mineralized tissues (i.e., pyridinoline [Pyr] and dehydrodihydroxylysinonorleucine [deH‐DHLNL]). This study shows that it is feasible to monitor Pyr and DHLNL collagen cross‐links spatial distribution in mineralized tissues. The spectroscopic parameter established in this study may be used in FTIRI analyses, thus enabling the calculation of relative Pyr/DHLNL amounts in thin (∼5 μm) calcified tissue sections with a spatial resolution of ∼7 μm.