Viable microorganisms are present in subglacial waters and sediment-laden ice beneath John Evans glacier in the Canadian high Arctic. The Bacterial communities resident in three subglacial samples were examined by amplifying 16S rRNA genes extracted from community DNA and from axenic isolates. Restriction fragment length polymorphism analysis of 341 clones produced 153 operational taxonomic units (OTUS), of which 25 dominant OTUS were sequenced. A subglacial water sample yielded Betaproteobacteria (25% of clones, particularly Comamonadaceae), Bacteroidetes (23%, particularly Flavobacterium) and Actinobacteria (14%). A second water sample had 51%Betaproteobacteria, 5%Bacteroidetes and no Actinobacteria, and a sediment sample was dominated by Betaproteobacteria (15%) and Bacteroidetes (38%). A collection of 158 morphologically distinct isolates was obtained on R2A agar using three incubation conditions: fully aerobic at 20°C or 4°C, or microaerobic at 20°C. A total of 52 isolate OTUs were defined, comprising Bacteroidetes (predominantly Flavobacterium isolated at 4°C), Betaproteobacteria (particularly Comamonadaceae), plus Actinobacteria and Alpha- and Gammaproteobacteria not detected as clones. Otherwise, the clone library and isolate collection results were quite comparable and supported earlier molecular studies at this site. Although additional undescribed diversity likely exists in these samples, combining culture-based results with molecular analysis increased the observed bacterial diversity and confirmed previous observations at this glacier and others.