The inhabitants of New Guinea and its outlying islands have played an important role in the human history of the Pacific region. Nevertheless, the genetic diversity, particularly of pre-colonial communities, is still understudied. Here we present the ancient genomes of 42 individuals from Papua New Guinea (PNG). The ancient genomic results of individuals from Watom Island (Bismarck Archipelago) and the south and northeastern coasts of PNG are contextualized with new (bio-) archaeological data. The individuals' accelerator mass spectrometry (AMS) dates span 2,500 years of human habitation, and our results demonstrate the influences of different dispersal events on the genetic make-up of ancient PNG communities. The oldest individuals show an unadmixed Papuan-related genetic signature, whereas individuals dating from 2,100 years before present carry varying degrees of an East-Asian-related contribution. These results and the inferred admixture dates suggest a centuries-long delay in genetic mixture with local communities after the arrival of populations with Asian ancestry. Two geographically close communities on the South Coast, AMS dated to within the past 540 years, diverge in their genetic profiles, suggesting differences in their interaction spheres involving groups with distinct ancestries. The inferred split time of these communities around 650 years before present coincides with intensified settlement activity and the emergence of regional trade networks.