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The Colombian paso horse, the most important horse breed in Colombia, performs specific and particular gaits (paso fino, trocha, and Colombian trot), which display different footfall patterns and stride frequencies. The breed has been selected for gait and conformation for more than 50 years and we hypothesize that this selection has led to kinematic differences of the gaits that can be explained by different genetic variants. Hence, the aims of the study were: 1. To identify if there are any differences in the kinematic and genetic variants between the Colombian paso horse's gaits. 2. To evaluate if and how much the gait differences were explained by the nonsense mutation in the DMRT3 gene and 3. To evaluate these results for selecting and controlling the horses gait performance. To test our hypotheses, kinematic data, microsatellites and DMRT3 genotypes for 187 Colombian paso horses were analyzed. The results indicated that there are significant kinematic and DMRT3 differences between the Colombian paso horse's gaits, and those parameters can be used partially to select and control the horses gait performance. However, the DMRT3 gene does not play a major role in controlling the trocha and the Colombian trot gaits. Therefore, modifying genes likely influence these gaits. This study may serve as a foundation for implementing a genetic selection program in the Colombian paso horse and future gene discovery studies for locomotion pattern in horses., Competing Interests: Gabriella Lindgren is co-inventor on a granted patent concerning commercial testing of the DMRT3 mutation: A method to predict the pattern of locomotion in horses. PCT EP12 747 875.8. European patent registration date: 2011-05- 05, US patent registration date: 2011-08-03. Miguel Novoa Bravo is personal of the company Genética Animal de Colombia Ltda. This funder provided support in the form of research materials, infrastructure, funding for research travel, and salary (after January the first 2018) for author MNB, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section. There are no further patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.