Bacteria have been isolated from virtually every environment on Earth. The Antarctic continent is no exception. In this extremely cold and dry environment bacteria have colonised various refugia and have evolved a number of strategies for coping with the extreme physico-chemical fluctuations they are exposed to within the environment. These psychrophilic adaptations include cold adapted proteins and lipids which are interest for biotechnology in areas such as frozen foods, agriculture and cryogenic storage. One type of cold adapted protein of particular interest is the antifreeze protein (AFP) for its recrystalisation inhibition and thermal hysteresis activity. It was first isolated from Antarctic fish in the 1970, but has since been found in plants, fungi, insects and bacteria. Over 800 bacterial isolates were cultured from lakes of the, Vestfold Hills, Larsemann Hills and MacRobertson Land, Antarctica. Approximately 87% were Gram negative rods. A novel AFP assay designed for high-throughput analysis in Antarctica, demonstrated putative activity in 187 isolates. Subsequent SPLAT analysis (qualification assessment of recrystalisation inhibition activity) of the putative positive isolates showed 19 isolates with significant recrystalisation inhibition activity. These 19 isolates were cultured from five separate lakes with substantial physico-chemical differences. The 19 AFP active isolates were characterised, using amplified ribosomal DNA restriction analysis (ARDRA) and 16S rDNA sequencing, as predominantly belonging to genera from the a- and y-Proteobacteria, although they were more prominent in the gamma subdivision. One of these isolates (213, Halomonas sp.) was shown as dominant within its community by DGGE analysis, indicating a possible selective advantage for AFP active bacteria. This is the first report of the phylogenetic distribution of AFP activity within bacteria, thus providing information which could enable future bacterial AFP assessments to be aimed at specific taxonomic groups.