Background/Objectives: Mild autonomous cortisol secretion (MACS) can coexist with primary aldosteronism (PA). The purpose of our study was to evaluate whether (MACS) influences parameters analyzed during adrenal venous sampling (AVS) in patients with PA. Methods: Patients with PA from the SPAIN-ALDO Registry and the German Conn’s Registry with available 1 mg-dexamethasone suppression test (DST) and AVS were included. MACS was defined as a post-DST cortisol > 1.8 µg/dL in the absence of specific signs and symptoms of Cushing’s syndrome. Results: Two-hundred and twenty-five patients were included, 98 (43.6%) of whom had concomitant MACS. The mean age was 54 ± 10 years and 37.3% were women. AVS was performed by simultaneous catheterization of both adrenal veins and analysis of basal samples in 157 patients (69.8%), with both basal and post-ACTH samples in 15 patients (6.7%), and during continuous ACTH infusion in 53 patients (23.6%). AVS was considered technically unsuccessful in 40 cases (17.8%), suggesting unilateral secretion in 106 (47.1%) and bilateral secretion in 79 (35.1%). We did not find significant differences in the percentage of unilateral and bilateral results, cortisol, corrected aldosterone, or selectivity indices in the dominant and non-dominant veins, nor in the lateralization index or the contralateral suppression index between patients with and without MACS. They also had similar rates of surgical treatment and biochemical and clinical response. Conclusions: Although pathophysiological reasoning suggests that MACS could hinder AVS identification of unilateral forms of PA, our data suggest that such interference, if it exists, is of moderate clinical relevance.