The first two mitochondrial genomes of marine diatoms were previously reported for the centric Thalassiosira pseudonana and the raphid pennate Phaeodactylum tricornutum. As part of a genomic project, we sequenced the complete mitochondrial genome of the freshwater araphid pennate diatom Synedra acus. This 46,657 bp mtDNA encodes 2 rRNAs, 24 tRNAs, and 33 proteins. The mtDNA of S. acus contains three group II introns, two inserted into the cox1 gene and containing ORFs, and one inserted into the rnl gene and lacking an ORF. The compact gene organization contrasts with the presence of a 4.9-kb-long intergenic region, which contains repeat sequences. Comparison of the three sequenced mtDNAs showed that these three genomes carry similar gene pools, but the positions of some genes are rearranged. Phylogenetic analysis performed with a fragment of the cox1 gene of diatoms and other heterokonts produced a tree that is similar to that derived from 18S RNA genes. The introns of mtDNA in the diatoms seem to be polyphyletic. This study demonstrates that pyrosequencing is an efficient method for complete sequencing of mitochondrial genomes from diatoms, and may soon give valuable information about the molecular phylogeny of this outstanding group of unicellular organisms.