We determined the spatial and temporal variation of phytoplankton biomass and diversity in 10 fortnightly sampling journeys verifying five sampling sites in El Peñol-Guatapé reservoir located in Antioquia, Colombia. In situ and ex situ physical and chemical variables were measured and phytoplankton samples were taken. Richness was dominated by Chlorophyta and biomass by Dinophyta. This variable was higher in the transition zone while in the riverine zone it showed average values, the lowest were registered in the lacustrine zone. Diversity was lower in the riverine zone, but it increased considerably in the transition zone and in the lacustrine zone where it showed similar values. At the vertical scale, biomass and diversity did not vary widely. Biomass showed a differential response to the effect of precipitation changes and water levels in the reservoir zones. Due to the low variability in environmental conditions, diversity was homogeneous time-wise. These results suggest that factors related to the hydrodynamic such as precipitation and water level resulting from the dam operation and inflow have a slight influence on the temporal variations of phytoplankton biomass and structure. There was a direct relationship between these attributes and physical variables such as turbidity and nutrients concentration at spatial scale while at the vertical scale, mixing patterns and the influence of the wind explained the absence of the vertical gradient of biomass and phytoplankton structure.