Here we report the complete sequence of the mitochondrial (mt) genome of the necrotrophic phytopathogenic fungus Sclerotinia borealis, a member of the order Helotiales of Ascomycetes. The 203,051 bp long mtDNA of S. borealis represents one of the largest sequenced fungal mt genomes. The large size is mostly determined by the presence of mobile genetic elements, which include 61 introns. Introns contain a total of 125,394 bp, are scattered throughout the genome, and are found in 12 protein-coding genes and in the ribosomal RNA genes. Most introns contain complete or truncated ORFs that are related to homing endonucleases of the LAGLIDADG and GIY-YIG families. Integrations of mobile elements are also evidenced by the presence of two regions similar to fragments of inverton-like plasmids. Although duplications of some short genome regions, resulting in the appearance of truncated extra copies of genes, did occur, we found no evidences of extensive accumulation of repeat sequences accounting for mitochondrial genome size expansion in some other fungi. Comparisons of mtDNA of S. borealis with other members of the order Helotiales reveal considerable gene order conservation and a dynamic pattern of intron acquisition and loss during evolution. Our data are consistent with the hypothesis that horizontal DNA transfer has played a significant role in the evolution and size expansion of the S. borealis mt genome.