UNLABELLED: There are only about 7,100 adolescent and adult cheetahs (Acinonyx jubatus) remaining in the wild. With the majority occurring outside protected areas, their numbers are rapidly declining. Evidence-based conservation measures are essential for the survival of this species. Genetic data is routinely used to inform conservation strategies, e.g., by establishing conservation units (CU). A commonly used marker in conservation genetics is mitochondrial DNA (mtDNA). Here, we investigated the cheetah's phylogeography using a large-scale mtDNA data set to refine subspecies distributions and better assign individuals to CUs. Our dataset mostly consisted of historic samples to cover the cheetah's whole range as the species has been extinct in most of its former distribution. While our genetic data largely agree with geography-based subspecies assignments, several geographic regions show conflicting mtDNA signals. Our analyses support previous findings that evolutionary forces such as incomplete lineage sorting or mitochondrial capture likely confound the mitochondrial phylogeography of this species, especially in East and, to some extent, in Northeast Africa. We caution that subspecies assignments solely based on mtDNA should be treated carefully and argue for an additional standardized nuclear single nucleotide polymorphism (SNP) marker set for subspecies identification and monitoring. However, the detection of the A. j. soemmeringii specific haplogroup by a newly designed Amplification-Refractory Mutation System (ARMS) can already provide support for conservation measures. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10592-022-01483-1.

BIOPOLIS Program in Genomics Biodiversity and Land Planning CIBIO Campus de Vairão 4485‑661 Vairão Portugal

Breeding Centre for Endangered Arabian Wildlife Sharjah United Arab Emirates

CE3C Centre for Ecology Evolution and Environmental Changes and CHANGE Global Change and Sustainability Institute Departamento de Biologia Animal Faculdade de Ciências Universidade de Lisboa 1749 016 Lisboa Portugal

Central European Institute of Technology University of Veterinary Sciences Brno Brno Czech Republic

Centro de Investigação Em Biodiversidade e Recursos Genéticos CIBIO InBIO Laboratório Associado Campus de Vairão Universidade Do Porto Vairão Portugal

Department of Animal Genetics University of Veterinary Sciences Brno Czech Republic

Department of Behavioral and Cognitive Biology University of Vienna Djerassiplatz 1 1030 Vienna Austria

Faculdade de Psicologia Universidade de Lisboa Alameda da Universidade 1649 013 Lisboa Portugal

Institute for Ecology Evolution and Diversity Goethe University Max von Laue Straße 13 60438 Frankfurt am Main Germany

Kölner Zoo AG Riehler Straße 173 50735 Cologne Germany

Konrad Lorenz Institute of Ethology Savoyenstraße 1 1160 Vienna Austria

LOEWE Centre for Translational Biodiversity Genomics Senckenberganlage 25 60325 Frankfurt am Main Germany

Mohitban Society No 91 Moghaddas Ardebili str Tehran Iran

Natural History Museum Central Research Laboratories Burgring 7 1010 Vienna Austria

Opel Zoo von Opel Hessische Zoostiftung Königsteinerstrasse 35 61476 Kronberg im Taunus Germany

Research Institute of Wildlife Ecology University of Veterinary Medicine Savoyenstraße 1 1160 Vienna Austria

Rex Foundation White Rock House 2 Whipsnade Road LU6 2NB Dunstable UK

School of Life Sciences University of KwaZulu Natal Durban South Africa

South African National Biodiversity Institute National Zoological Garden 232 Boom Street 0002 Pretoria South Africa

Wildlife Conservation Society New York NY USA

Zoological Institute Russian Academy of Sciences Saint Petersburg Russia

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