The first millennium AD witnessed profound cultural shifts in the Mediterranean region, marked by the rise and fall of the Western Roman Empire and the emergence of the Byzantine Empire. In the quest for a comprehensive understanding of human mobility, I employ a multidisciplinary approach, combining ancient DNA analysis, archaeological, and historical data. This study aims to unearth the genetic imprints of human mobility and diversity within coastal and inland regions of Anatolia, unveiling population dynamics across centuries and illuminating migration and settlement patterns. I analyze a dataset comprising ancient genomes encompassing the Mediterranean region, including Asia Minor, to shed light on historical connections both within and beyond the ecoregions of the Anatolian Peninsula, extending to Western Eurasia.Our findings underscore the intricate population structure and regional connectivity within Anatolia, which commenced prior to the Roman period. Systematic analyses of the data reveal interactions beyond Anatolia, highlighting that there was no massive population replacement in the region during this era. Northern western Anatolia exhibits a diverse gene pool, influenced by both eastern and western contributions, while central, western, and southern Anatolia's populations remained closely clustered until the end of the Roman Era. Later during Byzantine period, a few Western Mediterranean outliers emerge within the local population structure, which remains largely unaffected. The southern eastern ecoregion presents a distinct gene pool with affinities to Persian populations, demonstrating that the interaction sphere extended beyond western influences, aligning with historical records.