Adolescence is an important phase for the structural and functional development of the brain. The immaturity of adolescent brain development is associated with high susceptibility to exogenous disturbances, including alcohol. In this study, the acquisition of conditioned place preference (CPP) in adolescent mice by alcohol (2 g/kg) and the parvalbumin-positive interneurons (PV+ interneurons), oligodendrocyte lineage cells (OPCs), and myelination in the medial prefrontal cortex (mPFC) were assessed. We aim to determine the age- and subregional-specificity of the effects of alcohol. Alcohol (2 g/kg) was injected intraperitoneally on even days, and saline was injected intraperitoneally on odd days. The control group received a continuous intraperitoneal injection with saline. Differences in alcohol-induced CPP acquisition were assessed, followed by immunohistochemical staining. The results showed a pronounced CPP acquisition in 4- and 5-week-old mice. In the mPFC, there were reduced PV+ interneurons and OPCs in 3-week-old mice and reduced oligodendrocyte numbers in 4-week-old mice. The 5-week-old mice showed impaired myelination and a decrease in the number of PV+ interneurons, mature oligodendrocytes, and OPCs in the mPFC. Since the alterations in 5-week-old mice are more pronounced, we further explored the mPFC-associated subregional-specificity. In the alcohol-exposed mice, the oligodendrocyte numbers were decreased in the anterior cingulate cortex (ACC), PV+ interneuron numbers were declined in the prelimbic cortex (PL), and the number of oligodendrocytes, PV+ interneurons, and OPCs was also decreased with impaired myelination in the infralimbic cortex (IL). Our data suggest that adolescent alcohol exposure notably affected the acquisition of CPP, myelin formation, and the counts of PV+ interneurons, mature oligodendrocytes, and OPCs in the mPFC in 5-week-old mice. Also, the IL subregion was the worst-affected subregion of the mPFC in alcohol-exposed 5-week-old mice. It reveals that the effects of alcohol on adolescence and its mPFC myelination show obvious age- and subregional-specificity.