Objectives Debate persists regarding the biological makeup of European Ottoman communities settled during the expansion of the Ottoman Empire during the 16th and 17th centuries, and the roles of conversion and migration in shaping demography and population history. The aim of this study was to perform an assessment of the biological affinities of three European Ottoman series based on craniometric data. Materials and Methods Craniometric data collected from three Ottoman series from Hungary and Romania were compared to European and Anatolian comparative series, selected to represent biological affinity representative of historically recorded migration and conversion influences. Sex‐separated samples were analyzed using D2‐matrices, along with principal coordinates and PERMANOVA analyses to investigate biological affinities. Discriminant function analysis was employed to assign Ottoman individuals to two potential classes: European or Anatolian. Results Affinity analyses show larger than expected biological differences between males and females within each of the Ottoman communities. Discriminant function analyses show that the majority of Ottoman individuals could be classified as either European or Anatolian with a high probability. Moreover, location within Europe proved influential, as the Ottomans from a location of more geopolitical importance (Budapest) diverged from more hinterland communities in terms of biological affinity patterns. Discussion The results suggest that male and female Ottomans may possess distinct population histories, with males and females divergent from each other in terms of their biological affinities. The Ottoman communities appear diverse in terms of constituting a mix of peoples from different biological backgrounds. The greater distances between sexes from the same community, and the differences between communities, may be evidence that the processes of migration and conversion impacted individual people and groups diversely.