In recent years, mitochondrial DNA (mtDNA) typing has become established as a powerful tool for forensic investigations. The sequencing of mtDNA is now a standard laboratory procedure for the examination of degraded casework samples of human origin, with unique advantages over nuclear DNA profiling systems. Nonetheless, the use of mtDNA in non-human forensic science has only recently gained attention, with applications in the profiling of ‘silent witnesses’ of crimes, traceability of food products and control of illegal trade of endangered species or drugs, among others. This review briefly describes some important features of mtDNA with relevance for forensic testing in different taxonomic groups and suggests appropriate laboratorial and analytic procedures to avoid erroneous interpretations. It is imperative to merge a solid knowledge of mitochondrial genetics with rigorous methods of error prevention in order to permit the full approval of non-human mtDNA profiling in routine forensic work.