More than two decades of archaeological research at the site of Sagalassos, in southwest Turkey, resulted in the study of the former urban settlement in all its features. Originally settled in late Classical/early Hellenistic times, possibly from the later fifth century BCE onwards, the city of Sagalassos and its surrounding territory saw empires come and go. The Plague of Justinian in the sixth century CE, which is considered to have caused the death of up to a third of the population in Anatolia, and an earthquake in the seventh century CE, which is attested to have devastated many monuments in the city, may have severely affected the contemporary Sagalassos community. Human occupation continued, however, and Byzantine Sagalassos was eventually abandoned around 1200 CE. In order to investigate whether these historical events resulted in demographic changes across time, we compared the mitochondrial DNA variation of two population samples from Sagalassos (Roman and Middle Byzantine) and a modern sample from the nearby town of Ağlasun. Our analyses revealed no genetic discontinuity across two millennia in the region and Bayesian coalescence-based simulations indicated that a major population decline in the area coincided with the final abandonment of Sagalassos, rather than with the Plague of Justinian or the mentioned earthquake.Belgian Programme on Interuniversity Poles of Attraction grant: (IAP 07/09, http://iap-cores.be/); University of Leuven grant: (GOA 13/04); KU Leuven BOF Centre of Excellence Financing on ‘Centre for Archaeological Sciences 2–New methods for research in demography and interregional exchange’; Research Foundation Flanders (FWO) grants: (Projects G.0562.11, G.0637.15); Leverhulme Trust (UK) grant:(F/00212/AM); Institute of History of Leiden University