With the incorporation of more fossil evidence, the co-existence of archaic (“Homo erectus-like”) and derived (“modern-like”) dental morphological patterns were highlighted in East Asian specimens spanning from the mid-Middle to early Late Pleistocene. The possible presence of multiple human lineages was suggested but not fully understood. Using micro-CT techniques and a landmark-free geometric morphometric approach, we investigated premolar root structural organization and morphological variation of enamel-dentine junction (EDJ) of Chinese Middle Pleistocene hominins, and compared our results with a number of fossil and modern human groups. Our analyses highlighted an evolutionary trend of premolar structure from the early Middle Pleistocene to recent times. For the root structure, we found an evolutionary trend of shape simpliܪcation along the Homo lineage examined here, and detected erectus-like signals in one late Middle Pleistocene specimen from China. For the EDJ morphology, we found a trend toward crown base reduction, symmetric occlusal EDJ outline and higher EDJ topography, but also observed different evolutionary trajectories of the EDJ shape in Asian fossil hominins. Moreover, this study brings insights into the taxonomy/phylogeny of a few late Middle Pleistocene specimens (generally categorized as “archaic H. sapiens”) whose evolutionary placement was in debate. Considering the substantial differences, the morphological deviation between these samples cannot be simply interpreted as biogeographical variation of a single lineage. A scenario of geographical isolation, persistence of archaic lineages and possible admixture among populations during the late Middle Pleistocene is discussed.