Compositional changes of the bacterioplankton assemblages in four high altitude middle Atlas lakes were monitored over a complete seasonal cycle using combinations of culture-dependent and molecular approaches. Viable bacterial numbers varied between seasons, with the lowest numbers recorded in the winter and the highest in the summer in all four lakes. Also, bacterial occurrences were found to be strongly correlated with water temperature in all the four sites, i.e., Lake Aoua (0.88), Ifreh (0.59), Hechlef (0.77) and Affourgagh (0.79) during the study period. Standard microbiological characterization of bacteria isolates from the lakes revealed majority (68%) to be Gram positive. Seasonal variations in the microbial assemblages among the lakes were also validated by restriction fragment length polymorphism (RFLP). Shifts in microbial assemblages, especially of the fecal indicator bacteria appeared to also be influenced by differences in the morphometric and watershed characteristics among the lakes. This study further reveals the need to employ combinations of methodological approaches, including taxonomic, physiological and molecular methods to adequately delineate and fully understand the ecology of microbial assemblages in extreme environments.