Molecular techniques are powerful tools that can address many research problems in insect ecology. Mitochondrial DNA (mtDNA) is a widely used molecular marker. It is easy to use and has favorable biological properties, such as near-neutrality, lack of recombination, and a clock-like evolutionary rate. However, there are some issues involved when using mtDNA data in population genetics, species delimitation, and estimating the evolutionary history of populations and species. Exceptions to the simplicity of mitochondrial inheritance and other limitations include small effective population sizes, maternal inheritance, and complex evolutionary processes. Combining mtDNA and nuclear DNA markers can improve the power of molecular data to test phylogenetic and phylogeographic hypotheses. We review the applications of mtDNA in insect ecology and conclude that a better understanding of the properties of mitochondria is essential for the application of mtDNA.