The study on mitochondrial DNA (mtDNA) is a hot field in recent molecular biology. With some special characteristics, mitochondrial DNA as an ideal genetic marker has been successfully applied to the research of population and evolutionary biology of fish. Many scientific achievements on mtDNA in fish have been made, however, a number of issues need to be further studied and explored. In the present paper, the research progresses on the complete mtDNA sequences, the structures and characteristics of mtDNA, the mtDNA control region structures, and mtDNA polymorphisms and detecting methods are introduced. Furthermore,we review the applications in the recent years in the fields of systematic evolution, hybridization and introgression, biogeography and population genetics of fish.