Purpose: Gait variability is a determinant of qualified locomotion and is useful for monitoring the effects of therapeutic interventions. The aim of this study was to compare gait variability and symmetry in trained individuals with transtibial (TT) amputation and transfemoral (TF) amputation. Methods: The design of this study was planned as observational. Eleven individuals with TF amputation, 14 individuals with TT amputation, and 14 healthy individuals (HI) were evaluated with a motorized treadmill. The mean step length, the step length variability, an ambulation index, and the time on each foot (stance phase symmetry) of participants were recorded. Results: There were differences between the three groups in the residual/non-preferred limb (RNp) step length (p = 0.031), the intact/preferred (IP) limb step length variability (p = 0.001), the RNp step length variability (p < 0.001), the time on each foot (p < 0.001), and the ambulation index score (p < 0.001). There was a similarity between the groups (TF, TT, HI) in IP limb step lengths (p = 0.127) and duration of prosthesis usage since amputation in individuals with lower limb loss (p = 0.224). Conclusions: This study provided basic data about gait variability and symmetry in individuals with traumatic lower limb loss. The results of the study showed that the variability of gait increased with the level of loss, and individuals with TT amputation showed partially equivalent performance with the healthy group. Similarities in gait characteristics may have resulted from effective prosthetic usage or effective gait rehabilitation.