Bacterial population changes were investigated in the monomictic Lake Estanya by combining microscopic analysis and two molecular methods involving the amplification of 16S rDNA genes using primers for the domain Bacteria and subsequent restriction fragment length polymorphism (PCR-RFLP) and denaturing gradient gel electrophoresis (PCR-DGGE). Both approaches revealed the vertical distribution of predominant microbial morphotypes and phylotypes in both holomictic and stratified periods, respectively, and showed that variations in structure and composition of bacterial populations are occurring in this lake as a function of depth and time. Through principal component analysis (PCA), these shifts could be related to different physicochemical parameters with temperature, oxygen concentration, and the incident light being of paramount importance as structuring variables. Comparison of RFLP and DGGE profiles by scoring similarities using the Jaccard coefficient and then building a multidimensional scaling map (MDS) showed equivalent results. Both techniques revealed that bacterial populations, present in the whole water column in the holomictic period, showed a high similarity with those located in the deeper part of the lake in the stratified period, evidencing that other factors, both biotic and abiotic, should also be considered as a force driving change in the composition of the bacterial community. Furthermore, DGGE analysis showed that sequences from prominent bands were affiliated to members of four major phyla of the domain Bacteria: Cyanobacteria, Bacteroidetes, Proteobacteria, and Actinobacteria, most of which corresponded to heterotrophic bacterial populations involved in carbon, sulfide, and nitrogen biogeochemical cycles, which were indistinguishable under the light microscope. © 2007 Springer Science+Business Media, LLC.