Even though running performance and positional profiles in football are well described, amputee football (AF) has different characteristics of the movement, pitch dimensions, and time played. There is a gap in the scientific literature about positional profiles based on running performance in AF. This study aimed to investigate the differences between positions, the influence of the amputation level or defect of the lower limb (LD), the differences in running performance between halves, and the relationship with the final match result. Thirteen AF National Team players were monitored by Global Navigation Satellite System (GNSS), tracking 24 official international matches for 17 months. Values of top speed, peak acceleration, peak deceleration, average distance per minute, sprint mean speed, GPS load per minute, inertial load per minute (Gs load/min), number of sprints per minute, and a number of impacts per minute were analyzed for defenders (DEF), midfielders (MID) and forwards (FOR). Additional factors analyzed were amputation level (below the knee, low amputation—LA or over the knee, high amputation—HA or defect of the lower limb—LD) and the match's final result. Midfielders had significantly higher running performance parameters compared to other positions (greater top speed than DEF (+ 0.3 m/s;p <0.001) and FOR (+ 0.2 m/s;p =0.045), greater peak acceleration and deceleration than DEF (+ 0.5 m/s2for both measures;p <0.001) and FOR (+ 0.4 and + 0.3 m/s2;p <0.001 andp =0.036, respectively), greater GPS load/minute than DEF (+ 0.2 load/min;p =0.001) and FOR (+ 0.3 load/min;p <0.001), greater Gs load per minute than DEF (+ 2.7 load/min;p <0.001) and FOR (+ 1.8 load/min;p <0.001), greater number of impacts per minute than DEF (+ 0.2 n/min;p <0.001) and FOR (+ 0.2 n/min;p <0.001). Players with LD had significantly higher running performance than those with LA or HA. In the match's second half, a decrease in running performance was registered. The trend of running more when losing could be observed—AF players had higher running parameters in lost matches, but the differences were not statistically significant. Further research complied with contextual game analysis is needed to assess the running performance of AF players deeply.