Introduction <p>Localizing the source of adrenocorticotropic hormone (ACTH) in patients with ACTH-dependent Cushing’s syndrome (CS) poses significant challenges. There is a pressing need for novel imaging modalities that provide both anatomical delineation and functional confirmation of corticotropinomas.</p> Methods <p>This study utilized a modified form of desmopressin (mDesmo), an agonist of the V1b receptor, which is overexpressed in tumoral corticotrophs. After modification, radionuclide ⁶⁸Ga was chelated to produce a new radiopharmaceutical for positron emission tomography (PET) imaging. A total of 234 patients with CS were screened and 30 underwent [⁶⁸Ga]Ga-mDesmo imaging. Patient demographics and hormonal levels (plasma cortisol and ACTH) were recorded, and the diagnostic accuracy of contrast-enhanced MRI (CE-MRI) and [⁶⁸Ga]Ga-mDesmo was compared, with histopathological evidence of a corticotropinoma serving as the reference standard.</p> Results <p>The cohort comprised 10 males (33%, CI: 16–50%) and 20 females (67%, CI: 50–84), with a mean age of 33.5 ± 13.5 years. Of 30 patients with CS, 24 patients had treatment naïve Cushing’s disease (CD) of which 17 (71%, CI: 53–89%) had microadenomas and 7 (30%, CI: 12–48%) had macroadenoma; 4 (13%, CI: 0–25%) patients had ectopic Cushing syndrome (ECS). CE-MRI identified lesions in 16 patients (67%, CI: 48–86%), while [⁶⁸Ga]Ga-mDesmo successfully identified corticotropinomas in all 24 patients (100%, CI: 86–100%). In patients with microadenomas, CE-MRI had a diagnostic accuracy of 53% (CI: 29–77%), whereas [⁶⁸Ga]Ga-mDesmo achieved 100% (CI: 86–100%). There was no uptake of [⁶⁸Ga]Ga-mDesmo in the pituitary of patients with ECS.</p> Conclusion <p>This study demonstrates that [⁶⁸Ga]Ga-mDesmo imaging is an effective in vivo molecular imaging technique for delineating corticotropinomas, surpassing the accuracy of CE-MRI and BIPSS, regardless of adenoma size.</p> Trial registration number <p>CTRI/2022/08/044615 dated 03/08/2022.</p>