This study determined the complete mitochondrial (mt) genome sequence of the canine heartworm, Dirofilaria immitisThe complete nucleotide sequence for the mitochondrial genome of Dirofilaria immitis is available from the EMBL database under the Accession Number AJ537512., and compared its structure, organization and other characteristics with Onchocerca volvulus and other secernentean nematodes. The D. immitis mt genome is 13814 bp in size and contains 36 of the 37 genes typical of metazoan organisms, and lacks the ATP synthetase subunit 8 gene. All of the genes are transcribed in the same direction. For the entire genome, the nucleotide contents are ∼55% (T), ∼19% (each for A and G) and ∼7% (C), which is very similar to those of the protein-coding genes. In the latter genes, most (∼69%) third codon positions have a T, but rarely (∼1–9%) have an A or a C. The C content (8–12%) is higher at the first and second codon positions compared with the third position (∼1%). These nucleotide biases have a significant effect on the codon usage patterns and, thus, on the amino acid composition of the proteins. The mt genome organization of D. immitis is essentially the same as that of O. volvulus, but is distinctly different from other secernentean nematodes sequenced thus far. Irrespective of transpositions of transfer RNA (trn) genes and the non-coding, AT-rich region, there are 4 gene- or gene block-translocations between the mt genome of D. immitis and those of Caenorhabditis elegans, Ascaris suum and the 2 human hookworms, Ancylostoma duodenale and Necator americanus. For D. immitis, the 22 trn genes have secondary structures typical of other secernentean nematodes, and possess a TV-replacement loop instead of a TΨC arm and loop. Like O. volvulus, the mt trnK and trnP of D. immitis use the anticodons CUU and AGG, whereas in other nematodes, UUU and UGG are employed, respectively. Also, the secondary structures of the 2 ribosomal RNA (rrn) genes are similar to the models for other nematodes. Overall, the availability of the complete D. immitis mt genome sequence provides a resource for future studies of the comparative mt genomics and of the population genetics and/or phylogeny of parasitic nematodes.