We describe a non-invasive method of determining the subspecies identity of common chimpanzees (Pan troglodytes), based on subspecies-specific sequence differences in the mitochondrial genome. This procedure involves the extraction of DNA from hair, the amplification of a short (410 base pair (b.p.)) segment of the non-coding displacement loop (D-loop) by the polymerase chain reaction (PCR), and subspecies identification based on rapid allele-specific oligonucleotide (ASO) probe dot-blot typing. This approach will contribute to: (i) the colony-level management of captive chimpanzees by enabling managers to recognize hybrids between subspecies and minimize outbreeding depression; (ii) the recognition of inappropriately matched individuals in comparative behavioural and experimental studies; and (iii) forensic questions surrounding the origin of illegally traded animals.