Background: Arteriovenous fistula (AVF) is the preferred vascular access for hemodialysis but often fails to mature. AVF maturation requires venous dilation to allow increases in blood flow. The required venous dilation is likely affected by its microstructure. We hypothesized that the preoperative collagen fiber configuration index for the venous medial layer may determine venous dilation and hence AVF maturation. Methods: Veins were harvested from 84 kidney failure patients undergoing AVF creation Surgery at the University of Alabama at Birmingham. These vein samples were obtained from near the anastomosis, where the vein and artery were surgically connected. They were formalin fixed, paraffin embedded, and sectioned into 5µm thin sections. The second harmonic generation (SHG) signals in these sections were acquired and analyzed for anisotropy index (AI) and orientation angle (OA). AI ranged from 0 (random fiber network) to 1 (completely aligned fiber network). OA ranged from 0o (parallel to lumen) to 90o (perpendicular to lumen). The fiber configuration index (FCI) was defined as the product of AI and sin(OA). AVF duplex ultrasound was performed post-op at 6weeks. Maturation was defined as the ability for dialysis at blood flow ≥ 300 ml/min for at least 6 dialysis sessions in 1 month and within 6 months post-op. Results: AVF maturation failure occurred in 15.5 % of these patients. The 6-week AVF blood flow was positively associated with the FCI (per 0.1 unit difference in FCI: Δ blood flow = 131 ml/min; 95% CI, 8 to 254 ml/min; p=0.038). The FCI of clinically matured AVFs was significantly higher than that of non-matured AVFs (0.13±0.07 vs. 0.08±0.05, p=0.02). Conclusions: Using the novel fiber configuration index, we have characterized venous medial collagen fiber organization in kidney failure patients. The FCI values appear to be associated with the likelihood of AVF maturation.