Yarns manufactured by non-conventional spinning methods have mechanical properties that are different from those of the conventional ring spun yarns. This provides difficulties in optimizing successive processes such as weaving or knitting processes. Rotor spun yams have a specific fiber arrangement and thus an intrinsic internal structure that deviates from those of ring spun yarns due to the different twist insertion mechanism. This research reports on the 3-dimensional structural characteristics of the rotor spun yarns. We used the tracer fiber method in combination with the 2-dimensional refractive images, which enabled to perform the 3-dimensional analysis of yarn structure by the image processing technology. Results showed that the fiber trajectories of the rotor spun yarn were of its own characteristics. The constituent fibers are concentrated mostly around the radial position of half the yarn radius, whereas the fiber locations spread in a relatively narrow range. Internal structure of the rotor spun yarns remained unchanged in the low or middle level of twists. In a high twist level, however, the yarn structure changed significantly according to the twist, which could be confirmed though the migration factor and orientation angle distribution. Increasing twists resulted in a more hairy yarn, however the hair length became shorter.