Racehorses competing in short (i.e., ‘sprinters’), middle- or longer-distance (i.e., ‘stayers’) flat races are assumed to have natural variation in locomotion; sprinters having an innately shorter stride than stayers. No study has objectively tested this theory. Here, racehorses (n = 421) were categorised as sprinters, milers or stayers based on known race distance (n = 3269 races). Stride parameters (peak length and frequency) of those racehorses were collected from prior race-pace training sessions on turf (n = 2689; ‘jumpout’, n = 1013), using a locomotion monitoring device. Pedigree information for all 421 racehorses was extracted to three-generations. In training, sprinters had a shorter stride of higher frequency and covered consecutive furlongs faster than stayers (p < 0.001). Relatively short or longer stride did not predict race success, but stayers had greater race success than sprinters (p < 0.001). Peak stride length and frequency were moderately heritable (h2 = 0.15 and 0.20, respectively). In conclusion, differences in stride were apparent between sprinters and stayers (e.g., shorter stride in sprinters) during routine training, even after accounting for their pedigree. Objective data on stride characteristics could supplement other less objectively obtained parameters to benefit trainers in the appropriate selection of races for each individual racehorse.