Background: Peripheral artery disease (PAD) impacts over 200 million people worldwide. It is traditionally linked to atherosclerotic calcification, or intima arterial calcification (IAC). Recent findings emphasize the role of medial arterial calcification (MAC). Current studies have identified some factors in MAC and IAC, however, a comprehensive understanding of their distinct features and pathogenesis is lacking. This study addresses this gap by examining tibial arteries from PAD patients post-amputation. We characterized the calcification using microCT and histology. Methods: Human tibial arteries were obtained from PAD patients post amputation, and segmented into posterior, anterior, and peroneal arteries. The arteries were dissected, fixed in 4% paraformaldehyde, and stained with iodine. High-resolution microCT images were captured using a Scanco μCT40, and 3D models were reconstructed by ImageJ and Slicer3D. The samples were then processed for histological analysis, including Hematoxylin and eosin (H&E) stain and Alizarin red staining. Results: The study design is shown in Fig. A, featuring the serial dissection of a posterior artery as an example. The microCT scan protocol is designed for imaging continuous sections under a specific orientation. The 3D models enable a comprehensive assessment of calcification and quantification for distinct categorization, and the peroneal artery showed the least calcified lesions (*P<0.05, ***P<0.001; Fig. B & C). MAC/IAC or co-existing lesions were characterized by cross-sectional images and H&E staining. The results indicated a predominance of MAC in the highly calcified group, comprising over 98% of the entire tissue (Fig. D & E). Conclusions: We characterized calcified lesions in tibial arteries by microCT and histology. Calcification categories were determined through 3D models. Histology and cross-section images enabled the assessment and quantification of MAC/IAC or co-existing lesions.