Functional recovery is the primary goal of therapeutic intervention in neuromuscular rehabilitation. The purpose of this study was to perform a segmental kinematic analysis using both planar angles computation and a tridimensional (3D) reconstruction of the rat hindlimb, regarding the morphology and the movement of each segment. Seven rats were evaluated for natural overground walking, and motion capture of the right hindlimb was collected with an optoeletronic system while the animals walked in the track. 3D biomechanical analyses were carried out and hip, knee, ankle, and metatarsophalangeal joint angular displacements were calculated. For flexion/extension, the knee joint and toe segment were statistically different between planar and 3D analysis, with the toe segment performing less extension at initial contact (IC) and the amplitude during swing phase for the knee being larger. During abduction/adduction, all hip joint parameters were statistically different except at IC and toe-off (TO) instants, the planar angles being higher than the 3D angles. In the horizontal plane, significant differences were found for ankle peaks of rotation, with increased results for the planar angles. In conclusion, a comparison between planar and 3D segmental kinematic analysis using a tridimensional reconstruction of the rat hindlimb demonstrated that different joints have different motion patterns within motion planes, probably related with physiological constraints and muscle actions. A major indication of the need for an anatomical reference frame kinematic analysis is supported by the knowledge that neuromuscular diseases are related to important clinical signs or motor deficits that should be observed, qualified, and quantified.