Fear conditioning is a type of learning that is essential for animals to avoid a danger and has been studied in model animals. In mammals, the amygdala plays essential roles in fear conditioning. In teleost, the medial zone of the dorsal telencephalon (Dm) has been postulated to be a homolog of the mammalian amygdala based on both neuroanatomical and functional studies. However, Dm is a broad area in the dorsal telencephalon and the neural circuitry mediating the amygdala functions has yet to be explored. Here we identify the neuronal circuitry that is essential for both active avoidance and Pavlovian fear conditioning by a genetic approach using the model vertebrate zebrafish. We performed large-scale gene trap and enhancer trap screens and generated transgenic fish that expressed Gal4FF, a synthetic Gal4 transcription activator, in specific regions and neuronal circuits in the brain. Then we crossed these brain-specific Gal4FF transgenic fish lines with UAS-neurotoxin lines to inhibit the activity of the Gal4FF-expressing neurons. We found that, when the activity of a subpopulation of neurons in Dm was inhibited, the fish showed deficits in both active avoidance and Pavlovian fear conditioning. These neurons are glutamatergic, and have projections to other brain areas, including the hypothalamus, preoptic area, and entopeduncular nucleus. This finding should provide an insight into understanding evolutionarily conserved fear circuits in vertebrates.