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Objective: To investigate the intra-individual variability of the plasma aldosterone concentration (PAC) and the plasma renin concentration (PRC) and explore its impact on screening for primary aldosteronism (PA). Methods: This cross-sectional study enrolled patients with PA and patients with essential hypertension (EH) who were admitted to the First Affiliated Hospital of Chongqing Medical University from July 2018 to December 2024. Clinical and biochemical data, including PAC and PRC, were collected. The intra-individual coefficient of variation (CV) and percentage difference (PD) were calculated. The rates of PA diagnosis missed were further analyzed. Results: In total, 431 patients with PA [mean age 48.2 years; 192 males (44.5%)] and 495 patients with EH [mean age 49.6 years; 198 males (40.0%)] were included. In the PA group, the intra-individual CVs for PAC, PRC, and the aldosterone-to-renin ratio (ARR) were 21.3%, 36.9%, and 39.5%, respectively, and the corresponding mean PDs were 23.1%, 38.5%, and 43.5%. In the EH group, the intra-individual CVs for PAC, PRC, and ARR were 23.5%, 33.5%, and 32.5%, respectively, and the mean PDs were 25.7%, 36.1%, and 36.3%, respectively. Due to intra-individual variability, 10.2% (44/431) of PA patients may be missed during screening, 70.5% (31/44) of whom exhibit a history of hypokalemia and/or adrenal nodules. Conclusions: Plasma aldosterone and renin levels display considerable intra-individual variability, which may compromise the accuracy of PA screening. Repeat testing is recommended for patients with an initial negative screening result who have a history of hypokalemia and/or adrenal nodules.