Ritual tooth ablation, the intentional removal of teeth, is a highly visible form of body modification that can signal group identity and mark certain life events, such as marriage. The widespread occurrence of the practice in Asia appears to have begun in the Neolithic period and in some areas, such as Taiwan, continued until the ethnographic present. We aim to use a biocultural approach to investigate the significance of tooth ablation in Indonesia and Vanuatu during the maritime expansion of Austronesian-speaking groups ca. 3500-2000 years ago. Here we assess the presence and patterns of tooth ablation in four prehistoric skeletal assemblages from eastern Indonesia (Pain Haka, Melolo, Lewoleba and Liang Bua) and one from Vanuatu (Uripiv). Despite the relatively small sample sizes, it was found that individuals from all the sites displayed tooth ablation. The Indonesian populations had ablation patterns that involved the maxillary lateral incisors and canines and the individuals from Uripiv had the central maxillary incisors removed. We suggest that the distribution of tooth ablation in eastern Indonesia provides strong evidence that this practice was an important ritual process associated with the early expansion of Austronesian-speaking populations in the region. The identification of tooth ablation at the site of Uripiv is the earliest example of the practice in the Pacific Islands and was either a Southeast Asian tradition brought by Austronesian settlers, was introduced later from Near Oceania, or was an indigenous development in Vanuatu. A similar pattern of tooth ablation (the removal of central maxillary incisors) has been documented in ethnographic reports of northern Vanuatu tribes. We argue that the practice could possibly be a ritual passed through the generations since the early settlement of Vanuatu.