Aim The associated flood events to floodplain of many rivers in the world affect the composition and structure of aquatic biota due the water levels variation can ensure ecological integrity of associated wetlands. This study describes the spatial and temporal variation in the macrophyte assemblage in the Santo Tomás wetland during seasonally flood-pulsed of the Magdalena River (North of Colombia). Methods For eight months, between 2017 and 2018 samplings were carried out in three stations for composition and abundance of aquatic macrophytes. Spatio-temporal pattern of richness was estimated with Chao methodology and vegetation cover was calculated using Kruskal-Wallis and U Mann-Whitney tests. A range abundance curve was used for species dominance between samplings and stations. A non-metric multidimensional scaling (nMDS) was used to analyze the Spatio-temporal distribution, Canonical Correspondence Analysis (ACC) was performed to relate the physicochemical variables to the species composition. Results A total of 24 species of aquatic plants distributed in 23 genera and 15 families were registered in the transects. The most abundant species (% of vegetation cover) were Ipomoea aquatica (19%), followed by Ludwigia helminthorrhiza (14%) and Eichhornia azurea (13%). The most frequent life form was free-floating, followed by the emergent one. The Spatio-temporal changes and the highest values of richness and vegetation cover in the Santo Tomás wetlands occurred during the filling and high waters period. L. helminthorrhiza, E. azurea, Pistia stratiotes, Neptunia oleracea, I. aquatica, Salvinia auriculata, and Hymenachne amplexicaulis were the most dominant species. The quality water of Santo Tomás Wetlands showed spatial and temporal variations during flooding pulse and some physicochemical variables such as organic matter (COD, BOD5), pH, depth, ammonia, and fecal coliforms were related to macrophyte community composition Conclusions Spatial and temporal changes of aquatic plants in Santo Tomás wetlands were related to the flooding pulse of Magdalena River.