Little is known about the viability of eukaryotic microorganisms preserved in icy regions. Here we report on the diversity of microbial eukaryotes in ice samples derived from four Pyrenean glaciers. The species composition of eukaryotic communities in these glaciers is unknown mostly because of the presence of a multi-year ice cap, and it is not clear whether they harbor the same populations. The recent deglaciation of these areas is allowing an easy access to glacial layers that correspond to the Little Ice Age although some isolated deposits are attributed to previous glacial cycles. In this study, we use molecular 18S rRNA-based approaches to characterize some of the microbial eukaryotic populations associated with Pyrenean glaciers. Firstly, we performed a chemical and microscopical characterization of ice samples. Secondly, molecular analyses revealed interesting protist genetic diversity in glaciers. In order to understand the microbial composition of the ice samples the eukaryotic communities resident in the glacial samples were examined by amplifying community DNA and constructing clone libraries with 18S rRNA primers. After removal of potential chimeric sequences and derreplication of identical sequences, phylogenetic analysis demonstrated that several different protists could be identified. Protist diversity was more phylum rich in Aneto and Monte Perdido glaciers. The dominant taxonomic groups across all samples (> 1 % of all sequences) were Viridiplantae and Rhizaria. Significant variations in relative abundances of protist phyla between higher and lower glaciers were observed. At the genus level, significant differences were also recorded for the dominant genera Chloromonas, Raphidonema , Heteromita , Koliella and Bodomorpha. In addition, protist community structure showed significant differences between glaciers. The relative abundances of protist groups at different taxonomic levels correlated with the altitude and area of glaciers and with pH.