Closed cryoconite holes (CCHs) are small aquatic ecosystems enclosed in glacier surface ice, and they collectively contribute substantial aquatic habitat to inland Antarctica. We examined the morphology, geochemistry and bacterial diversity of 57 CCHs, spread over seven sites, located on five glaciers, covering a range of latitudes, elevations and distance from open seawater. Isotopes confirmed glacial ice as the initial water source, with water chemistry evolving through freeze concentration and photosynthetic processes to have conductivities ranging from <0.005 to >4 mS cm−1 and pH from <5 to >11. Nitrate concentrations were more elevated in inland, higher altitude sites. Bacterial communities were characterized by Automated Ribosomal Intergenic Spacer Analysis and high-throughput sequencing. The dominant phyla were Cyanobacteria, Bacteroides, Proteobacteria and Actinobacteria. CCH bacterial communities predominantly grouped by geographic location, suggesting initial wind-borne inocula from local and regional sources play a role in structuring assemblages. However, multivariate multiple regression analysis indicated that internal CCH conditions also influenced community structure, particularly the ion content and pH of the liquid water. This highlights the importance of founder bacterial populations, isolation and water chemistry in the evolution of CCH bacterial communities.