Fish have colonized nearly all aquatic niches, making them an invaluable resource to understand vertebrate adaptation and gene family evolution, including the evolution of complex neural networks and modulatory neurotransmitter pathways. Among ancient regulatory molecules, the gaseous messenger nitric oxide (NO) is involved in a wide range of biological processes. Because of its short half-life, the modulatory capability of NO is strictly related to the local activity of nitric oxide synthases (Nos), enzymes that synthesize NO from L-arginine, making the localization of Nos mRNAs a reliable indirect proxy for the location of NO action domains, targets, and effectors. Within the diversified actinopterygian nos paralogs, nos1 (alias nnos) is ubiquitously present as a single copy gene across the gnathostome lineage, making it an ideal candidate for comparative studies. To investigate variations in the NO system across ray-finned fish phylogeny, we compared nos1 expression patterns during the development of two well-established experimental teleosts (zebrafish and medaka) with an early branching holostean (spotted gar), an important evolutionary bridge between teleosts and tetrapods. Data reported here highlight both conserved expression domains and species-specific nos1 territories, confirming the ancestry of this signaling system and expanding the number of biological processes implicated in NO activities.