AbstractThe Greenland Ice Sheet (GrIS) exports approximately 400 km³ of freshwater annually to downstream freshwater and marine ecosystems. These meltwaters originate in a wide range of well‐defined habitats that can be associated with very different physical environments within the ice sheet, ranging from oxygenated surface environments that are exposed to light and supplied with nutrients from atmospheric/aeolian sources to subglacial environments that are permanently dark, isolated from the atmosphere, and potentially anoxic. Hydrological conditions in the latter likely favor prolonged rock‐water contact. The seasonally evolving hydrological system that drains meltwaters from the GrIS connects these distinct microbial habitats and exports the microbes contained within them to downstream ecosystems. The microbial assemblages exported in glacier meltwater may have an impact on downstream ecosystem function and development. We explored how the seasonal development of a glacial drainage system influences the character of microbial assemblages exported from the GrIS by monitoring the seasonal changes in hydrology, water chemistry, and microbial assemblage composition of meltwaters draining from a glacier in southwest Greenland. We found that the microbial assemblages exported in meltwaters varied in response to glacier hydrological flow path characteristics. Whether or not meltwaters passed through the subglacial environment was the first‐order control on the composition of the microbial assemblages exported from the glacier, while water source (i.e., supraglacial or extraglacial) and subglacial residence times were second‐order controls. Glacier hydrology therefore plays a fundamental role in determining the microbial exports from glaciated watersheds.