SUMMARY: MitoFlex is a linux-based mitochondrial genome analysis toolkit, which provides a complete workflow of raw data filtering, de novo assembly, mitochondrial genome identification and annotation for animal High Throughput Sequencing data. The overall performance was compared between MitoFlex and its analogue MitoZ, in terms of protein-coding gene recovery, memory consumption and processing speed. AVAILABILITYAND IMPLEMENTATION: MitoFlex is available at https://github.com/Prunoideae/MitoFlex under GPLv3 license. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.