Background: The diagnosis of attention deficit hyperactivity disorder (ADHD) relies on history and observation, as no reliable biomarkers have been identified. In this study, we compared a large single diagnosis group of patients with ADHD (combined, inattentive, and hyperactive) to healthy controls using brain perfusion single-photon emission computed tomography (SPECT) imaging to determine specific brain regions which could serve as potential biomarkers to reliably distinguish ADHD. Methods: In a retrospective analysis, subjects ( n = 1,135) were obtained from a large multisite psychiatric database, where resting state (baseline) and on-task SPECT scans were obtained. Only baseline scans were analyzed in the present study. Subjects were separated into two groups - Group 1 ( n = 1,006) was composed of patients who only met criteria for ADHD with no comorbid diagnoses, while a control group ( n = 129) composed of individuals who did not meet criteria for any psychiatric diagnosis, brain injury, or substance use served as a non-matched control. SPECT regions of interests (ROIs) and visual readings were analyzed using binary logistic regression. Predicted probabilities from this analysis were inputted into a Receiver Operating Characteristic analysis to identify sensitivity, specificity, and accuracy. Results: The baseline ROIs and visual readings show significant separations from healthy controls. Sensitivity of the visual reads was 100% while specificity was >97%. The sensitivity and specificity of the post-hoc ROI analysis were both 100%. Decreased perfusion was primarily seen in the orbitofrontal cortices, anterior cingulate gyri, areas of the prefrontal cortices, basal ganglia, and temporal lobes. In addition, ROI analysis revealed some unexpected areas with predictive value in distinguishing ADHD, such as cerebellar subregions and portions of the temporal lobes. Conclusions: Brain perfusion SPECT distinguishes adult ADHD patients without comorbidities from healthy controls. Areas which were highly significantly different from control and thus may serve as biomarkers in baseline SPECT scans included: medial anterior prefrontal cortex, left anterior temporal lobe, and right insular cortex. Future studies of these potential biomarkers in ADHD patients with comorbidities are warranted., Competing Interests: TH is the president and principal owner of the Synaptic Space, a neuroimaging consulting firm. He is also CEO and Chairman of the Board of Neuro-Luminance Corporation, a medical service company. He is also president and principal owner of Dr. Theodore Henderson, Inc., a medical service company. He is also Vice-President of the Neuro-Laser Foundation, a non-profit organization. He is a member of and a former officer of the Brain Imaging Council Board of the Society of Nuclear Medicine and Molecular Imaging (SNMMI). Since 2017, he has served in the SNMMI Brain Imaging Outreach Working Group. Currently, he serves as president of the International Society of Applied Neuroimaging. TH has no ownership in, and receives no remuneration from, any neuroimaging company. No more than 5% of his income is derived from neuroimaging. DA is the sole owner of Amen Clinics, a group of nine neuropsychiatric clinics that perform brain SPECT imaging. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Amen, Henderson and Newberg.)