Simple SummaryTo understand mitochondrial genome fragmentation in bird lice, we sequenced the mitochondrial genomes of 17 species of bird lice in the families Menoponidae and Laemobothriidae. Of the 17 species, four species of Menoponidae have fragmented mitochondrial genomes, whereas the other 13 species of both Menoponidae and Laemobothriidae retain the typical single-chromosome mitochondrial genomes. Our phylogenetic analyses showed that mitochondrial genome fragmentation occurred multiple times independently in these two families. We also found derived mitochondrial minichromosomal characters shared between bird lice in the same genus and among different genera. We conclude that while mitochondrial genome fragmentation as a general feature does not unite all the parasitic lice that have this feature, each independent mitochondrial genome fragmentation event may produce shared derived mitochondrial minichromosomal characters that can be useful for resolving the phylogeny of parasitic lice at different taxonomic levels.AbstractMitochondrial (mt) genome fragmentation has been discovered in all five parvorders of parasitic lice (Phthiraptera). To explore whether minichromosomal characters derived from mt genome fragmentation are informative for phylogenetic studies, we sequenced the mt genomes of 17 species of bird lice in Menoponidae and Laemobothriidae (Amblycera). Four species of Menoponidae (Actornithophilussp. 1 ex [pied oystercatcher],Act.sp. 2 ex [masked lapwing],Austromenoponsp. 2 ex [sooty tern and crested tern],Myr.sp. 1 ex [satin bowerbird]) have fragmented mt genomes, whereas the other 13 species retain the single-chromosome mt genomes. The twoActornithophilusspecies have five and six mt minichromosomes, respectively.Aus.sp. 2 ex [sooty tern and crested tern] has two mt minichromosomes, in contrast toAus.sp. 1 ex [sooty shearwater], which has a single mt chromosome.Myr.sp. 1 ex [satin bowerbird] has four mt minichromosomes. When mapped on the phylogeny of Menoponidae and Laemobothriidae, it is evident that mt genome fragmentation has occurred multiple times independently among Menoponidae and Laemobothriidae species. We found derived mt minichromosomal characters shared betweenMyrsideaspecies, betweenActornithophilusspecies, and between and among different ischnoceran genera, respectively. We conclude that while mt genome fragmentation as a general feature does not unite all the parasitic lice that have this feature, each independent mt genome fragmentation event does produce minichromosomal characters that can be informative for phylogenetic studies of parasitic lice at different taxonomic levels.