Characterization of glutamatergic input to dorsal raphe (DR) serotonin (5-HT) neurons is crucial for understanding how the glutamate and 5-HT systems interact in psychiatric disorders. Markers of glutamatergic terminals, vGlut1, 2 and 3, reflect inputs from specific forebrain and midbrain regions. Punctate staining of vGlut2 was homogeneous throughout the mouse DR whereas vGlut1 and vGlut3 puncta were less dense in the lateral wing (lwDR) compared with the ventromedial (vmDR) subregion. The distribution of glutamate terminals was consistent with the lower miniature excitatory postsynaptic current frequency found in the lwDR; however, it was not predictive of glutamatergic synaptic input with local activity intact, as spontaneous excitatory postsynaptic current (sEPSC) frequency was higher in the lwDR. We examined the morphology of recorded cells to determine if variations in dendrite structure contributed to differences in synaptic input. Although lwDR neurons had longer, more complex dendrites than vmDR neurons, glutamatergic input was not correlated with dendrite length in the lwDR, suggesting that dendrite length did not contribute to subregional differences in sEPSC frequency. Overall, glutamatergic input in the DR was the result of selective innervation of subpopulations of 5-HT neurons and was rooted in the topography of DR neurons and the activity of glutamate neurons located within the midbrain slice. Increased glutamatergic input to lwDR cells potentially synergizes with previously reported increased intrinsic excitability of lwDR cells to increase 5-HT output in lwDR target regions. Because the vmDR and lwDR are involved in unique circuits, subregional differences in glutamate modulation may result in diverse effects on 5-HT output in stress-related psychopathology.