The Hawaiian Archipelago is a "biodiversity hotspot" where significant endemism among eukaryotes has evolved through geographic isolation and local topography. To address the absence of corresponding region-wide data on Hawaii's microbiota, we compiled the first 16S SSU rDNA clone libraries and cultivated bacteria from five Hawaiian lakes, an anchialine pool, and the Lō'ihi submarine volcano. These sites offer diverse niches over approximately 5000 m elevation and approximately 1150 nautical miles. Each site hosted a distinct prokaryotic community dominated by Bacteria. Cloned sequences fell into 158 groups from 18 Bacteria phyla, while seven were unassigned and two belonged in the Euryarchaeota. Only seven operational taxonomic units (each OTU comprised sequences that shared > or =97% sequence identity) occurred in more than one site. Pure bacterial cultures from all sites fell into 155 groups (each group comprised pure cultures that shared > or =97% 16S SSU rDNA sequence identity) from 10 Bacteria phyla; 15 Proteobacteria and Firmicutes were cultivated from more than one site. One hundred OTUs (60%) and 52 (33.3%) cultures shared <97% 16S SSU rDNA sequence identity with published sequences. Community structure reflected habitat chemistry; most delta-Proteobacteria occurred in anoxic and sulfidic waters of one lake, while beta-Proteobacteria were cultivated exclusively from fresh or brackish waters. Novel sequences that affiliate with an Antarctic-specific clade of Deinococci, and Candidate Divisions TM7 and BRC1, extend the geographic ranges of these phyla. Globally and locally remote, as well as physically and chemically diverse, Hawaiian aquatic habitats provide unique niches for the evolution of novel communities and microorganisms.