Markerless motion capture allows whole-body movements to be captured without the need for physical markers to be placed on the body. This enables motion capture analyses to be conducted in more ecologically valid environments. However, the influences of varied clothing on video-based markerless motion capture assessments remain largely unexplored. This study investigated two types of clothing conditions, “Sport” (gym shirt and shorts) and “Street” (unrestricted casual clothing), on gait parameters during overground walking by 29 participants at self-selected speeds using markerless motion capture. Segment lengths, gait spatiotemporal parameters, and lower-limb kinematics were compared between the two clothing conditions. Mean differences in segment length for the forearm, upper arm, thigh, and shank between clothing conditions ranged from 0.2 cm for the forearm to 0.9 cm for the thigh (p < 0.05 for thigh and shank) but below typical marker placement errors (1 – 2 cm). Seven out of 9 gait spatiotemporal parameters demonstrated statistically significant differences between clothing conditions (p < 0.05), however, these differences were approximately ten times smaller than minimal detectable changes in movement-related pathologies including multiple sclerosis and cerebral palsy. Hip, knee, and ankle joint angle root-mean-square deviation values averaged 2.6° and were comparable to previously reported average inter-session variability for this markerless system (2.8°). The results indicate that clothing, a potential limiting factor in markerless motion capture performance, would negligibly alter meaningful clinical interpretations under the conditions investigated.