Early adversity represents a main risk factor for the emergence of psychiatric disorders in adult life. In mice, postnatal days 2 to 14 constitute a critical period of vulnerability when stressors and environmental perturbations can alter the maturation of prefrontal circuits. Maternal separation (MS) has been well-established as a model to study the impact of early-life stress on neurodevelopment. MS during the critical period produces morpho-functional changes in the prefrontal cortex (PFC), accompanied by increases in depressive-like and anxiety behaviors. PFC descending circuits exert a top-down regulatory control over the dorsal raphe nucleus (DRN), the main source of forebrain serotonin (5-HT). Activation of PFC afferents in the DRN increases active stress-coping strategies and mood control. We studied whether MS during the critical period could modify the synaptic architecture of the developing PFC-to-DRN circuit. We analyzed the abundance of glutamatergic (vGLUT1/vGLUT2) and GABAergic (GAD2) synaptic afferents in the DRN after the last MS exposure (at day 15). Synaptic boutons were quantitatively analyzed using the high-resolution immunofluorescence technique Array Tomography. We find that MS increases the prefrontal innervations onto DRN neurons, without affecting either subcortical glutamate or GABA afferents. Our study indicates a selective developmental effect of stress on the PFC-to-DRN circuit, likely contributing to emotional vulnerability in the MS model.