Temporal and spatial changes of virioplankton abundance (VLP), chlorophyll-a (Chl-a) concentration, and some environmental variables, were assessed from October 2004 to April 2005 at four sampling sites in a high-mountain reservoir in the Colombian Andes. VLP ranged from 2.4-10.5×107 and 3.6- 6.5×107 VLP mL−1 in the samples from the photic zone and hypolimnion, respectively. Surface VLP showed a progressive increase from November to April in the limnetic zone, and until February in the littoral stations. This trend coincided with the gradual increase of the water column stratification, as well as the augment of the reservoir hydraulic volume. Principal components analysis showed a grouping of environmental (dissolved oxygen, pH, water temperature) and biological variables (VLP, Chl-a, bacterioplankton abundance and biomass) within the first component (26.4% of explained variance), associated to an increase of photosynthetic activity, as a potential supplement of organic substrates to heterotrophic viral hosts. High values of virus-to-prokaryote ratio (VPR), and a negative relationship between VLP and average biomass per prokaryotic cell, indicate strong viral control which is probably more intense on active and larger cells. These results provide a framework for understand the virioplankton responses to regional hydrological conditions and hydraulic behavior of this reservoir. The spatiotemporal scale of this study does not allow to confirm that viral dynamics is significantly affected by human activities causing potential alterations on nutrient cycling.