Walking deterioration is a common problem in people with multiple sclerosis (PwMS). However, there are only scarce data examining the contribution of specific neurological functional systems on gait performance in multiple sclerosis (MS). Therefore, the objective of the current study was to examine the differences in spatio-temporal parameters of gait according to the pyramidal, cerebellar and sensory functional systems. The cross-sectional study included 289 PwMS with mean disease duration of 8.0 (SD = 8.2) years. Spatio-temporal parameters of gait were studied using an electronic walkway. The sample pool was divided into six groups according to the scores of the pyramidal, cerebellar and sensory functional systems, derived from the expanded disability status scale data. Findings indicated that asymmetry of the step time and asymmetry of the single support were significantly elevated in the pyramidal group compared to the sensory group; 9.4 (SD = 10.6) vs. 3.1 (SD = 6.7), P value = 0.004; 9.3 (SD = 10.4) vs. 2.7 (2.1), P value = 0.001, respectively. Additionally, patients in the pure sensory group walked significantly faster with longer strides and less asymmetry compared to the pyramidal-cerebellar-sensory group. Moreover, patients in the sensory group walked with longer steps/strides and symmetry compared to the patients in the pyramidal-cerebellar group. This study confirms that pyramidal disorders are main contributors of gait impairments in the MS population. Furthermore, patients with sensory impairments have a relatively preserved gait pattern compared to patients affected by the pyramidal system.