Homo naledi displays a combination of features across the skeleton not found in any other hominin taxon, which has hindered attempts to determine its placement within the hominin clade. Using geometric morphometrics, we assess the morphology of the mandibular premolars of the species at the enamel-dentine junction (EDJ). Comparing with specimens of Paranthropus, Australopithecus and Homo (n = 97), we find that the H. naledi premolars from the Dinaledi chamber consistently display a suite of traits (e.g., tall crown, well-developed P 3 and p 4 metaconid, strongly developed P 3 mesial marginal ridge, and a P 3 > p 4 size relationship) that distinguish them from known hominin groups. Premolars from a second locality, the Lesedi Chamber, are consistent with this morphology. We also find that two specimens from South Africa, SK 96 (usually attributed to Paranthropus) and Stw 80 (Homo sp.), show similarities to the species, and we discuss a potential evolutionary link between H. naledi and hominins from Sterkfontein and Swartkrans. Homo naledi is a hominin species first described in 2015 based on remains from the Dinaledi Chamber in the Rising Star cave system in South Africa 1 , and subsequently from a second chamber in the cave, the Lesedi Chamber 2. The species presents a combination of features not found in any other taxon, and attempts to interpret its phylogenetic position within the hominin clade have proved difficult. There are remarkably modern features such as the morphology of the foot 3 , as well as the morphology of the wrist and the relative length of the thumb 4. However, the cranial capacity is small, both absolutely and relative to body size 1,2 , and there are primitive Australopithecus-like traits in the fingers 4 , upper and lower limbs 5,6 and pelvis 7. Studies of the dental evidence have likewise revealed a unique combination of features. The permanent postcanine dentition is characterized by small teeth that retain principal molar cusps, but seemingly lack accessory crown traits common in other African hominin groups 1,8. However, the deciduous dentition shows a number of derived Paranthropus-like traits 9 , and molar root metrics find similarities between H. naledi and South African Homo specimens SK 15 and SK 45, as well as KNM-ER 1805 from Koobi Fora 10. Mandibular premolar morphology is particularly useful in studies of hominin taxonomy 11-15 , and initial descriptions suggest the P 3 of H. naledi is highly distinctive. The tooth is absolutely small in size, double rooted, fully bicuspid, and has a symmetrical occlusal outline, a combination of features suggested to be unique among the hominin fossil record 1. Recent morphometric studies of the mandibular premolar enamel-dentine junction (EDJ) demonstrate that this method has the potential to be a powerful tool in distinguishing between hominin taxa 16,17. We therefore aim to further investigate this distinctive premolar morphology at the EDJ.