The serotonin (5-HT) system is generally considered as a single modulatory system, with broad and diffuse projections. However, accumulating evidence points to the existence of distinct cell groups in the raphe. Here, we review prior evidence for raphe cell heterogeneity, considering different properties of 5-HT neurons, from metabolism to anatomy, and neurochemistry to physiology. We then summarize more recent data in mice and zebrafish that support a genetic diversity of 5-HT neurons, based on differential transcription factor requirements for the acquisition of the 5-HT identity. In both species, PET1 plays a major role in the acquisition and maintenance of 5-HT identity in the hindbrain, although some 5-HT neurons do not require PET1 for their differentiation, indicating the existence of several transcriptional routes to become serotoninergic. In mice, both PET1-dependent and -independent 5-HT neurons are located in the raphe, but have distinct anatomical features, such as the morphology of axon terminals and projection patterns. In zebrafish, all raphe neurons express pet1, but Pet1-independent 5-HT cell groups are present in the forebrain. Overall, these observations support the view that there are a number of distinct 5-HT subsystems, including within the raphe nuclei, with unique genetic programming and functions.