The dorsal raphe (DR) is a heterogeneous nucleus containing dopamine (DA), serotonin (5HT), γ-aminobutyric acid (GABA) and glutamate neurons. Consequently, investigations of DR circuitry require Cre-driver lines that restrict transgene expression to precisely defined cell populations. Here, we present a systematic evaluation of mouse lines targeting neuromodulatory cells in the DR. We find substantial differences in specificity between lines targeting DA neurons, and in penetrance between lines targeting 5HT neurons. Using these tools to map DR circuits, we show that populations of neurochemically distinct DR neurons are arranged in a stereotyped topographical pattern, send divergent projections to amygdala subnuclei, and differ in their presynaptic inputs. Importantly, targeting DR DA neurons using different mouse lines yielded both structural and functional differences in the neural circuits accessed. These results provide a refined model of DR organization and support a comparative, case-by-case evaluation of the suitability of transgenic tools for any experimental application.