Abstract The study of dental morphology can be a very useful tool to understand the origin and evolution of Neanderthals in Europe during the Middle Pleistocene (MP). At present, the earliest evidence, ca. 430 ka, of a pre-Neanderthal population in Europe is the hominin sample from Atapuerca-Sima de los Huesos (SH) that present clear dental affinities with Neanderthals while other penecontemporaneous populations, such as Arago or Mala Balanica, exhibit less Neanderthal traits. We present the morphometric study of the external and internal dental structures of eleven hominin dental remains recovered from the MP, ca. 240 ka, French site of Biache-Saint-Vaast (BSV). Our analyses place the BSV hominins within the MP group, together with SH, Fontana Ranuccio, Visogliano, Steinheim or Montmaurin, that show greater morphological affinities with Neanderthals. Moreover, we identified interpopulation variability in the expression of the enamel thickness trait, with BSV hominins sharing the unique combination of thin and thick pattern in the premolars and molars with the SH population. These results further support the coexistence of two or more populations in Europe during the MP that reflect the population and settlement of human groups suggested by the Central Area of Dispersals of Eurasia (CADE) and sink and source model.