The composition of the eukaryotic community and the three-dimensional structure of diverse phototrophic microbial mats from two hot springs in Iceland (Seltun and Hveradalir geothermal areas) were explored by comparing eukaryotic assemblages from microbial mats. Samples were collected in July 2007 from 15 sampling stations along thermal and pH gradients following both hot springs. Physicochemical data revealed high variability in terms of pH (ranging from 2.8 to 7), with high concentrations of heavy metals, including up to 20 g Fe/l, 80 mg Zn/l, 117 mg Cu/l, and 39 mg Ni/l at the most acidic sampling points. Phylogenetic analysis of 18S rDNA genes revealed a diversity of sequences related to several taxa, including members of the Bacillariophyta, Chlorophyta, Rhodophyta, and Euglenophyta phyla as well as ciliates, amoebae, and stramenopiles. The closest relatives to some of the sequences detected came from acidophilic organisms, even when the samples were collected at circumneutral water locations. Electron microscopy showed that most of the microecosystems analyzed were organized as phototrophic microbial mats in which filamentous cyanobacteria usually appeared as a major component. Deposits of amorphous minerals rich in silica, iron, and aluminium around the filaments were frequently detected.