Studies of periventricular white matter injury (PWMI) in preterm infants suggest the involvement of the transient cortical subplate zone. We studied the cortical wall of non‐cystic and cystic PWMI cases and controls. Non‐cystic PWMI corresponded to diffuse white matter lesions, the predominant injury currently detected by imaging. Glial cell populations were analyzed in post‐mortem human frontal lobes from very preterm 24–29 postconceptional weeks (pcw) and preterm infants (30–34 pcw) using immunohistochemistry for glial fibrillary acidic protein (GFAP), monocarboxylate transporter 1 (MCT1), ionized calcium‐binding adapter molecule 1 (Iba1), CD68 and oligodendrocyte lineage (Olig2). Glial activation extended into the subplate in non‐cystic PWMI but was restricted to the white matter in cystic PWMI. Two major age‐related and laminar differences were observed in non‐cystic PWMI: in very preterm cases, activated microglial cells were increased and extended into the subplate adjacent to the lesion, whereas in preterm cases, an astroglial reaction was seen not only in the subplate but throughout the cortical plate. There were no differences in Olig2‐positive pre‐oligodendrocytes in the subplate in PWMI cases compared with controls. The involvement of gliosis in the deep subplate supports the concept of the complex cellular vulnerability of the subplate zone during the preterm period and may explain widespread changes in magnetic resonance signal intensity in early PWMI.