Serotonin neurons in the dorsal raphe defined by the expression of transcription factor Pet1, were specifically targeted and sorted using a microfluidic sorting device called On-Chip Sort. Sorted cells were then sequenced using 10x Genomics and Chromium single cell sequencing. We defined up to 14 different Pet1 neurons in the dorsal raphe, broadly split based on the expression of genes related to neurotransmitter synthesis and release including Tph2 (Serotonin synthesis), Gad2/1 (GABA synthesis), and Slc17a8 (packaging glutamate into vesicles). Each cluster expressed a unique set of differentially expressed g-protein coupled receptors, ion channels, transcription factors and other genes, however one cluster, cluster 12, was the most transcriptionally unique cluster. Notably, cluster 12, also referenced here as the Met/Pet1 group, uniquely expressed multiple neuropeptide receptors including ATP/ADP receptor P2ry1, Gastric inhibitory peptide (Gipr), Tachykinin receptor 3 (Tacr3), and Relaxin receptor 1 (Rxfp1). Also notable was the unique lack of expression of Orexin, Catecholamine, and Histamine receptors expressed in every other Pet1 group. The lack of expression of these receptors, which play a role in serotonin’s involvement in generalized arousal and the circadian rhythm, suggest that cluster 12 may have an opposing role in arousal than other Pet1 neurons. To gain a greater understanding of this cluster, we targeted them specifically using the intersectional expression of P2ry1-cre and Pet1-Flpe. Intersectional, dual recombinase alleles allow for fluorescent protein expression within cells expressing both cre and Flp. We found that P2ry1-Pet1 neurons were specifically clustered under the aqueduct, in a region called the caudal dorsal raphe. In addition, this subpopulation of serotonin neurons was the predominant ventricular projecting population of Pet1 neurons, and had fibers coating the walls of the third and lateral ventricles, including near the adult stem cell population in the subventricular zone. Slice electrophysiological recordings also showed that P2ry1-Pet1 neurons were less excitable than other neurons in the dorsal raphe, and had a longer latency to action potential. Future directions will focus on determining the function of P2ry1 Pet1 neurons, as well as characterizing the transcriptional heterogeneity of other raphe regions and through development.