Objectives Central venous sampling (CVS) with corticotropin-releasing hormone (CRH) stimulation is a crucial technique in evaluating adrenocorticotropic hormone (ACTH)-dependent Cushing’s syndrome (CS). We evaluated central venous sampling (CVS) and magnetic resonance imaging (MRI) findings in predicting the localization and lateralization of pituitary microadenomas. Methods We analyzed 29 patients with CS who underwent CVS with CRH stimulation and examined with MRI retrospectively. Catheterization to central sinuses was successfully performed in 26 patients. Three patients with variant anatomy or inability to cannulate were diagnosed with CD after examination of pathology. Results After CVS, among 26 patients, 23 patients were determined to have CD (88.4%) and 2 (7.7%) patients were diagnosed with ectopic ACTH syndrome. One patient was diagnosed with CD postoperatively. While the sensitivity of the CVS was 95.6%, sensitivity of the preoperative pituitary MRI was lower (69.5%). Also, the negative predictive value ratio was higher in CVS than in MRI (66% versus 22%). Diagnostic accuracy in the lateralization of the tumor was high as in CVS as in MRI (76.4% versus 73.9%). Conclusion Central venous sampling with higher sensitivity in the localization of pituitary microadenoma, also has approximately similar diagnostic accuracy in lateralizing the tumor with MRI.