Mixotrophy seems to be widespread among phytoplankton, but whether this strategy is more relevant in oligotrophic lakes remains unclear. Here, we tested the hypothesis that the relative abundance of mixotrophic flagellates in lakes increases along an elevational gradient paralleling increasingly oligotrophic conditions. For this purpose, 12 lakes located between 575 and 2796 m above sea level were sampled in summer and fall to include two different seasonal windows in phytoplankton dynamics and environmental conditions. The degree of mixotrophy in phytoplankton was estimated in tracer experiments using fluorescently-labeled bacteria and done with composite samples collected in the euphotic zone and in samples obtained from the chlorophyll-a maximum. The results indicated the existence of a positive trend particularly in summer in the relative abundance of mixotrophic flagellates with elevation, however, this trend was not linear, and exceptions along the elevational gradient were found. Changes in the relative abundance of mixotrophic flagellates were related with significant changes in water transparency, DOC and phosphorus concentrations, as well as in bacterial and flagellate abundance. Overall, our results reveal that the harsh growth conditions found in oligotrophic high mountain lakes favor a mixotrophic trophic strategy among phytoplankton.