We recently demonstrated patterned stratum corneum maturation and skin barrier formation during fetal development in rodents and rabbit. The presence of skin patterning in these mammals led us to predict patterned barrier formation during human infant development. Here we extend our mammalian study and demonstrate patterned stratum corneum development and skin barrier formation in the pre-term human infant. Surprisingly, we show initiation of human barrier regionally as early as 20–24 wk gestational age (22–26 wk menstrual age), bringing barrier formation close to the time of periderm disaggregation. We use the mouse model to show that patterns of periderm disaggregation mirrors barrier formation. Periderm disaggregation follows and recapitulates barrier pattern, suggesting a relationship between the processes. This work reveals regional patterning in skin maturation and barrier formation in the human infant and demonstrates that initiation of human skin barrier formation in utero coincides with the current lower limit of viability of the pre-term infant.