Summary1. In October 2004, plankton samples were collected from six permanent lakes located between 4960 and 5440 m a.s.l. in the Mount Everest region (Nepal) to assess how spatial and local environmental factors affect natural bacterial community composition. Fingerprinting analysis of the bacterial 16S rRNA gene fragment was done by denaturing gradient gel electrophoresis (DGGE).2. The number of DGGE bands (range: 12–23) was not correlated with lake area or remoteness, but there was a strong negative correlation with the ratio of catchment to lake area (r = −0.826, P < 0.05), suggesting that hydraulic retention time affects the establishment of the bacterial community in these seepage lakes.3. Most dominant sequences belonged to Betaproteobacteria except in two lakes where members of Bacteroidetes made the largest relative contribution. Up to 81% of the phylotypes had high similarity (>98 to 100%) in partial 16S rRNA gene sequence to those reported from other alpine lakes and glaciers around the world, suggesting the presence of ‘cosmopolitan’ bacteria.4. An analysis based on dissimilarity matrices and the Mantel test revealed the existence of dissimilarities in bacterial community composition related to geographical distance over a small spatial scale (<6 km), but determined by local environmental constraints.5. Our results suggest that several bacterial phylotypes are ubiquitous in the freshwater aquatic realm, but taxon sorting by local environmental constraints is important.