The serotonin (5-HT) system is the target of multiple anxiolytics, including Buspirone, which is a partial agonist of the serotonin 1A receptor (5‑HT1A). Similarly, ligands of the serotonin 2A receptor (5-HT2A) were shown to alter anxiety level. The 5-HT1A and 2A receptors are widely expressed across the brain, but the target region(s) underlying the influence of those receptors on anxiety remain unknown. Interestingly, recent studies in human and non-human primates have shown that the 5-HT1A and 5-HT2A binding potential within the insular cortex (insula) are correlated to anxiety. As an initial step to define the function of 5‑HT transmission in the insula, we quantified the proportion of specific neuronal populations of the insula expressing 5‑HT1A or 5‑HT2A. We analyzed six neural populations, including two defined by their fast amino acid transmitter (excitatory or inhibitory), and four defined by their projections to different downstream targets. First, we found that more than 70% of glutamatergic neurons, and only 30% of GABAergic neurons express the 5‑HT1A. Second, 5-HT1A is highly expressed (~80%) in the insular populations projecting to two sub-nuclei of the amygdala (central and basolateral), as well as in the populations projecting to the rostral and caudal sections of the lateral hypothalamus (LH). Similarly, 70% of insular glutamatergic neurons and only 20% of insular GABAergic neurons contain 5-HT2A. Finally, the 5-HT2A is present in most of insula-amygdala and insula-LH projection neurons (>60%). These observations suggest that a majority of glutamatergic neurons can respond to 5-HT through 5-HT1A or 5‑HT2A in the insula, and that 5-HT directly affects a limited number of GABAergic neurons. This study defines a molecular and neuroanatomical map of the 5-HT system within the insular cortex, providing ground knowledge to identify the potential role of serotonergic modulation of selective insular populations in anxiety.