Objective To investigate the characteristics of efferent projections from 5‑hydroxytryptamine (5‑HT)‑ergic neurons in the dorsal raphe nucleus (DRN) in mice, and to explore the differences in anatomical distribution of 5‑HT‑ergic neurons that project to different brain regions in the DRN. Methods A specific anterograde tracing virus rAAV2/9‑Ef1α‑DIO‑mCherry was injected into the DRN of Sert‑Cre mice (n=3). After virus expression for four weeks, the mice underwent transcardiac perfusion, and the brains were removed and cut into 40 μm‑thick coronal slices. The whole‑slide immunofluorescence imaging scans were performed to observe the distribution of 5‑HT‑ergic neurons in the downstream projection brain regions of the DRN. The specific retrograde tracing viruses rAAV2/retro‑EF1α‑DIO‑BFP, rAAV2/retro‑EF1α‑DIO‑mCherry, and rAAV2/retro‑EF1α‑DIO‑EGFP were injected into the nucleus accumbens (NAc), central nucleus of the amygdala (CeA) and ventral tegmental area (VTA) of Sert‑Cre mice (n=3), respectively. After virus expression for four weeks, the mice underwent transcardiac perfusion and the brains were collected. Then, slices containing the NAc, CeA, VTA and DRN were prepared for observing the anatomical distribution of virus‑labeled 5‑HT‑ergic neurons in the DRN under a confocal fluorescence microscope. Results 5‑HT‑ergic neurons in the DRN extensively projected to the whole brain, including the piriform cortex, medial prefrontal cortex, NAc, basal forebrain, CeA, lateral sclerotic nucleus, lateral hypothalamus, and VTA. Notably, 5‑HT‑ergic neurons projecting to the NAc were mainly distributed in the dorsal central and lateral regions of the DRN, 5‑HT‑ergic neurons projecting to the CeA were mainly located in the dorsal lateral region of the DRN, and 5‑HT‑ergic neurons projecting to the VTA were mainly located in the ventral interfascicular region of the DRN. Conclusions The anatomical distribution of 5‑HT‑ergic neurons in the DRN that project to different brain regions are different, indicating the existence of relatively independent multiple parallel subsystems of 5‑HT‑ergic neurons within the DRN.