Gait recovery after stroke is a primary goal of rehabilitation, therefore it is imperative to develop technologies that accurately identify gait impairments after stroke. Markerless motion capture (MMC) is an emerging technology that has been validated in healthy individuals. Our study aims to evaluate the validity of MMC against an instrumented walkway system (IWS) commonly used to evaluate gait in stroke survivors. Nineteen participants performed three comfortable speed (CS) and three fastest speed (FS) walking trials simultaneously recorded with IWS and MMC system, KinaTrax (HumanVersion 8.2, KinaTrax Inc., Boca Raton, FL, USA). Pearson’s correlation coefficient and intraclass correlation coefficient (ICC (3,1), 95%CI) were used to evaluate the agreement and consistency between systems. Furthermore, Bland–Altman plots were used to estimate bias and Limits of Agreement (LoA). For both CS and FS, agreements between MMC and IWS were good to excellent in all parameters except for non-paretic single-limb support time (SLS), which revealed moderate agreement during CS. Additionally, stride width and paretic SLS showed poor agreement in both conditions. Biases eliminated systematic errors, with variable LoAs in all parameters during both conditions. Findings indicated high validity of MMC in measuring spatiotemporal gait parameters in stroke survivors. Further validity work is warranted.