This study aimed to examine the influence of RunScribe location (i.e. lace shoe vs heel shoe) on the accuracy of spatiotemporal gait characteristics during running by comparing data with a high-speed video analysis system at 1000 Hz. A total of 49 endurance runners performed a running protocol on a treadmill at comfortable velocity. Two systems were used to determine spatiotemporal parameters (i.e. contact time, flight time, step frequency, and step length) during running: high-speed video analysis at 1000 Hz and two different RunScribe placements (i.e. lace shoe vs heel shoe). The pairwise comparisons showed some between-system differences in both lace shoe (contact time: p = 0.009; step frequency: p = 0.001) and heel shoe (flight time: p = 0.006; step frequency: p = 0.010), although the effect sizes were small (effect size < 0.3 in all cases). The intraclass correlation coefficients revealed an almost perfect association between systems for contact time and flight time (intraclass correlation coefficient: 0.85–0.90), and step length and step frequency (intraclass correlation coefficient: 0.96–0.97), regardless of the RunScribe placement. Bland–Altman plots revealed that the lace shoe location yielded smaller systematic bias, random errors, and narrower limits of agreement for spatiotemporal parameters during running, except for SF, which had a higher accuracy in a heel shoe location. The results suggest that RunScribe is a valid system to measure spatiotemporal parameters during running on a treadmill according to a high-speed video analysis at 1000 Hz. In addition, the data indicate that the location of the RunScribe system (lace shoe vs heel shoe) plays an important role on the accuracy of spatiotemporal parameters. The lace shoe placement showed smaller systematic bias, random errors, and narrower limits of agreement for contact time, flight time, and step length, whereas the heel shoe placement was slightly more accurate for the step frequency.