Introduction: Several histologic features have been identified in the upper-extremity arteries and veins of patients with advanced chronic kidney disease, which may affect arteriovenous fistula maturation. However, it is unclear whether these chronic kidney disease vascular features are abnormal. Methods: We obtained upper-extremity arterial and venous specimens from 125 advanced chronic kidney disease patients undergoing arteriovenous fistula creation and from 15 control subjects. We quantified medial fibrosis, micro-calcification, and intimal hyperplasia with appropriate histology stains. We characterized medial collagen fiber configuration in second-harmonic-generation microscopy images for the fiber anisotropy index and the dominant fiber direction. Results: The advanced chronic kidney disease patients were significantly younger than control subjects (53 ± 14 years vs 76 ± 11 years, p < 0.001). After controlling for age, the chronic kidney disease patients had greater arterial medial fibrosis (69% ± 14% vs 51% ± 10%, p < 0.001) and greater arterial micro-calcification (3.03% ± 5.17% vs 0.01% ± 0.03%, p = 0.02), but less arterial intimal thickness (30 ± 25 µm vs 63 ± 25 µm, p < 0.001), as compared to control subjects. The anisotropy index of medial collagen fibers was lower in both arteries (0.24 ± 0.10 vs 0.44 ± 0.04, p < 0.001) and veins (0.28 ± 0.09 vs 0.53 ± 0.10, p < 0.001) in chronic kidney disease patients, indicating that orientation of the fibers was more disordered. The dominant direction of medial collagen fibers in chronic kidney disease patients was greater in the arteries (49.3° ± 23.6° vs 4.0° ± 2.0°, p < 0.001) and the veins (30.0° ± 19.6° vs 3.9° ± 2.1°, p < 0.001), indicating that the fibers in general were aligned more perpendicular to the lumen. Conclusion: Advanced chronic kidney disease is associated with several abnormalities in vascular histology and collagen fiber configuration. Future research is needed to investigate whether these abnormalities affect the maturation outcomes of arteriovenous fistulas.