Strains of Ophiostoma ulmi, O. novo-ulmi subsp. americana, O. novo-ulmi subsp. novo-ulmi and O. himal-ulmi were examined for optional introns/insertions within the following mitochondrial genes: small subunit RNA gene (rns), large ribosomal subunit gene (rnl) and the cytochrome oxidase subunit I gene (coxI). Insertions were noted in the rns and coxI genes in strains of O. ulmi, the less aggressive species, but absent in strains of the more aggressive O. novo-ulmi subsp. americana. Strains of all species examined had a group I intron present in the U11 region of the mitochondrial-rnl gene. In all but two strains of O. novo-ulmi subsp. americana, this rnl-U11 intron was about 1.5 kb in length whereas a 2.6 kb version of this element was present in all strains representing O. ulmi, O. novo-ulmi subsp. novo-ulmi, and Ophiostoma himal-ulmi. Irrespective of size, this intron based on RNA folds is a class IA1 group I intron and it encodes a putative ORF for the rps3 ribosomal protein. The size variation of the rnl-U11 intron was examined in detail for two strains of O. novo-ulmi subsp. americana and sequence data suggests the presence of a complex ORF within the 2.6 kb version of this intron; here a homing endonuclease-like gene has been inserted in frame and fused to the carboxyl-terminus of the putative rps3 coding region. The mitochondrial optional introns/insertions in combination with nuclear markers might be useful in distinguishing among the various species and subspecies of the O. ulmi s. lat. complex.