Atomic force microscopy (AFM) micro-indentation combined with high-resolution MALDI-FTICR mass spectrometry imaging enabled the characterization of mechanical and molecular differences between young and aged equine energy-storing (SDFT) and positional (CDET) tendons. The FM and IFM exhibited distinct molecular profiles and mechanical properties, with differences depending on tendon type and age group. Integrating mechanical mapping with chemical imaging therefore provides insight into the spatial organization and functional specialization of tendon matrix compartments.