Study Design Collagen fiber orientation analysis in moderately degenerated human cadaveric annulus fibrosus (AF) tissue samples. Objective Little is known about the changes in tissue architecture during early degeneration of intervertebral disks (IVDs). As collagen organization strongly affects the disk function, the objective of this study was to quantify the AF collagen orientation and its spatial distribution in moderately degenerated IVDs (Pfirrmann grade III). Methods AF tissue samples were dissected from four circumferential (anterior, left and right lateral, and posterior) and two radial (outer and inner) locations. Cryosections were imaged using Second Harmonic Generation microscopy, and the collagen fiber orientations per location were determined utilizing a fiber-tracking image analysis algorithm. Also, the proportionality between the fibers aligned in the primary direction versus other oriented fibers was determined. Results Mean collagen fiber angles ranged between 21 and 31 degrees for outer and 15 to 19 degrees for inner AF samples. Mean collagen orientations at circumferential locations were only significantly different from each other at inner anterior and lateral location. Similarly, fiber angles between the outer and inner AF were not significantly different except at the posterior location. Fiber orientation proportionality did not show large variations. Except for a significant difference in outer AF proportionality between posterior and lateral positions, no other differences were observed. Conclusion The results of this study provide the first quantitative evidence that the collagen fiber orientation of moderately degenerated disks exhibits a spatial rather than homogeneous distribution and typical collagen orientation gradients characterizing healthy IVDs are only partially retained.