The rostro-medial cortex of the mouse and rat, considered the functional homolog to the primate prefrontal cortex (PFC), is of growing importance for preclinical models of schizophrenia and other neurodevelopmental diseases for which symptoms typically emerge in adolescence and early adulthood. Therefore, in order to explore molecular mechanisms operating during these critical stages of PFC development, it will be important to develop an efficient gene delivery system for the PFC of juvenile animals. To this end, adeno-associated virus (AAV)-based systems are increasingly used in mice for targeted gene delivery in specific brain regions such as the hippocampus. Strikingly, there is very little literature on vector-mediated gene expression in the rostro-medial cortex. In addition, multiple AAV serotypes exist based on differences in their envelope capsid proteins. However, to date, the large majority of studies in the central nervous system (CNS) have utilized the AAV2 serotype. This is typically limited to a very focal transduction pattern and therefore is not ideal for the murine PFC, which occupies several square millimeters in the rostral hemisphere. Here, we introduce a protocol for efficient, AAV9-serotype-mediated gene delivery in juvenile (postnatal day 21) and young adult PFC, resulting in long-lasting transgene expression.