Elevated dispositional negativity (DN; i.e., neuroticism/negative emotionality) is associated with a range of deleterious outcomes, including mental illness. Yet, DN’s neurobiology remains incompletely understood. Prior work suggests that DN reflects heightened threat-elicited reactivity in the extended amygdala (EAc), a circuit encompassing the central nucleus (Ce) and the bed nucleus of the stria terminalis (BST), and that this association may be intensified for uncertain threat. We utilized a multi-trait, multi-occasion DN composite and neuroimaging assays of threat anticipation and perception to demonstrate that individuals with elevated DN show heightened BST activation during threat anticipation. Analyses revealed that DN is uniquely predicted by BST reactivity to uncertain threat. DN was unrelated to Ce activation during threat anticipation or EAc activation during ‘threatening’-face presentation. Follow-up analyses revealed that the threat paradigms are not interchangeable probes of EAc function. These observations lay the foundation for future studies necessary to determine causation and improve interventions.