Trade in ivory from extant elephant species namely Asian elephant (Elephas maximus), African savanna elephant (Loxodonta africana) and African forest elephant (Loxodonta cyclotis) is regulated internationally, while the trade in ivory from extinct species of Elephantidae, including woolly mammoth, is unregulated. This distinction creates opportunity for laundering and trading elephant ivory as mammoth ivory. The existing morphological and molecular genetics methods do not reliably distinguish the source of ivory items that lack clear identification characteristics or for which the quality of extracted DNA cannot support amplification of large gene fragments. We present a PCR-sequencing method based on 116 bp target sequence of the cytochrome b gene to specifically amplify elephantid DNA while simultaneously excluding non-elephantid species and ivory substitutes, and while avoiding contamination by human DNA. The partial Cytochrome b gene sequence enabled accurate association of ivory samples with their species of origin for all three extant elephants and from mammoth. The detection limit of the PCR system was as low as 10 copy numbers of target DNA. The amplification and sequencing success reached 96.7% for woolly mammoth ivory and 100% for African savanna elephant and African forest elephant ivory. This is the first validated method for distinguishing elephant from mammoth ivory and it provides forensic support for investigation of ivory laundering cases.