Serotonin neurotransmitter deficits are reported in suicide, major depressive disorder (MDD) and alcohol use disorder (AUD). To compare pathophysiology in these disorders, we mapped brain serotonin transporter (SERT), 5-HT, and 5-HT receptor binding throughout prefrontal cortex and in anterior cingulate cortex postmortem. Cases and controls died suddenly minimizing agonal effects and had a postmortem interval ≤24 h to avoid compromised brain integrity. Neuropathology and toxicology confirmed absence of neuropathology and psychotropic medications. For most subjects (167 of 232), a DSM-IV Axis I diagnosis was made by psychological autopsy. Autoradiography was performed in right hemisphere coronal sections at a pre-genual level. Linear model analyses included sex and age with group and Brodmann area as interaction terms. SERT binding was lower in suicides (p = 0.004) independent of sex (females < males, p < 0.0001), however, the lower SERT binding was dependent on MDD diagnosis (p = 0.014). Higher SERT binding was associated with diagnosis of alcoholism (p = 0.012). 5-HT binding was greater in suicides (p < 0.001), independent of MDD (p = 0.168). Alcoholism was associated with higher 5-HT binding (p < 0.001) but only in suicides (p < 0.001). 5-HT binding was greater in suicides (p < 0.001) only when including MDD (p = 0.117) and alcoholism (p = 0.148) in the model. Reported childhood adversity was associated with higher SERT and 5-HT binding (p = 0.004) in nonsuicides and higher 5-HT binding (p < 0.001). Low SERT and more 5-HT and 5-HT binding in the neocortex in depressed suicides is dependent on Axis I diagnosis and reported childhood adversity. Findings in alcoholism differed from those in depression and suicide indicating a distinct serotonin system pathophysiology.