The fungal order Ophiostomatales contains numerous species important in medical fields, agriculture, and forestry, and several species have had available mitogenome information. The nuclear genome of the entomopathogenic fungus Sporothrix insectorum has been reported, while its mitogenome remains unknown. Herein, we firstly described the mitogenome of S. insectorum RCEF 264 and then compared Ophiostomatales mitogenomes from both interspecific and intraspecific perspectives. The mitogenome of S. insectorum RCEF 264 was 31,454 bp in length, containing typical fungal mitochondrial genes plus rnpB. Four group I introns interrupted rnl and cox1. Phylogenetic analyses confirmed the placement of S. insectorum RCEF 264 in Ophiostomatales. Comparison of mitogenomes among seven Ophiostomatales species revealed conserved gene contents and a high synteny, although there were also some differences among them. Their mitogenomes showed more than two-fold variations (26.6-65.1 kb) in size, with a total of 37 intron insertional loci from 11 genes (1-25 introns per species). The sole intron shared by all species was an rps3-encoding intron in rnl (mL2450), and this intron-based phylogeny was highly consistent with those constructed using mitochondrial/nuclear genes, suggesting convergent evolution of this intron with Ophiostomatales species. The dendrogram based on presence/absence patterns at all intron loci was quite different from those based on mitochondrial/nuclear genes. Comparison of mitogenomes among two to three intraspecific individuals in Ophiostoma novo-ulmi subsp. novo-ulmi and Sporothrix schenckii revealed mitogenome size variations due to single-nucleotide polymorphisms (SNPs) and indels but without fluctuation of intron numbers for each species. This study greatly enhanced our understanding of mitogenome evolution in Ophiostomatales.