Over the last 10,000 years, Southeast Asia has seen a series of cultural transitions, many of which have left an impact onthe human genetic diversity of the region. The focus of this work is to investigate a series of demographic hypotheses presented by linguists, palaeoanthropologists, archaeologistsand geneticists from an ancient DNA approach. Ancient DNA research in thehot humid climate of Southeast Asia has been made possible by recent advancements in the field. Chapter 1 focusses primarily on the transition from hunter-gathering to farming in Southeast Asia. Debate has been recurrent for over a century, regarding the mode in which the transition to farming had occurred. The generally favoured model, the two-layermodel, posits that farmers from the north spread into Southeast Asia, bringing the farming technology. The alternative model is one of regional continuity; the hunter-gatherers of Southeast Asia themselves began farming, resulting from a spread of ideas rather than a spread and replacement of people. We generated genomes from hunter gatherers (8,000 –4,000 years old) and early farmers to people from historical times (4,000 –500 years old) and show that with the appearance of farming, people arrived that were genetically distinct from the hunter gatherers. These farmers had origins in the north, where rice and millet farming originated. Chapter 2 is a follow up study, bringing the total number of ancient and historical genomes from Southeast Asia to 60, with an additional 36 genomes from relevant present-day Southeast Asian populations. With this increased sample density, we show in more detail differences between early hunter-gatherers, migrations between Island and Mainland Southeast Asia, and demonstrate the genetic impact of human populations in the neighbouring regions –India and China. Chapter 3 is a small manuscript focussed on a single site at a single point in time: 15thCentury Kota Melaka, Malaysia. Under the Melaka Sultanate, the port town was an important trade site to the Indian Ocean, a link in the chain from East Asia to Europe. By 1511, the Portuguese had captured the city and build a fort –Kota Melaka. Beneath thefort,three individuals were buried together. On morphological grounds they were identified as ‘caucasoid’, a task made challenging due to the deterioration of the skeletons. Sequencing low coverage whole genomes revealed a vastly different story stretching across the Indian Ocean. While one man appears local, the other two individuals lacked Southeast Asian ancestry completely: one was a woman with origins in South India or Sri Lanka, and the other was a man from Southeast Africa. Combined with the style, location and dating of burials, they appear to have been royal companions or slaves to the Melaka Sultanate.The work in this thesis demonstrates both the viability of undertaking aDNA studies in hot and humid regions, and the resolution that ancient DNA research can provide to long standing questions