The diversity of highly active bacterial communities in cryoconite holes on three Arctic glaciers in Svalbard was investigated using terminal restriction fragment length polymorphism (T-RFLP) of the 16S rRNA locus. Construction and sequencing of clone libraries allowed several members of these communities to be identified, with Proteobacteria being the dominant one, followed by Cyanobacteria and Bacteroidetes. T-RFLP data revealed significantly different communities in holes on the (cold) valley glacier Austre Brøggerbreen relative to two adjacent (polythermal) valley glaciers, Midtre Lovénbreen and Vestre Brøggerbreen. These population compositions correlate with differences in organic matter content, temperature and the metabolic activity of microbial communities concerned. No within-glacier spatial patterns were observed in the communities identified over the 2-year period and with the 1 km-spaced sampling. We infer that surface hydrology is an important factor in the development of cryoconite bacterial communities.