Background Tuberculosis (TB) has contributed to a significant disease burden and economic loss worldwide. Given no gold standard for diagnosis, early identification of TB infection has been challenging. This study aimed to comparatively investigate the prevalence of TB across diagnostic approaches (sputum AFB, sputum culture, sputum genetic test, and chest x-ray) and geographical areas of Indonesia. Methods Participant demographic variables and TB screening test results were obtained from the Tuberculosis Unit, Health Research and Development Agency, Ministry of Health (HRDA-MoH). The prevalence of pulmonary TB in populations aged 15 years and over was calculated using TB cases as a numerator and populations aged 15 years and over as a denominator. Variations across geographical areas and diagnostic approaches were expressed as prevalence and 95% confidence interval (CI). Results A total of 67,944 records were reviewed. Based on bacteriological evidence, the prevalence of TB per 100,000 in Indonesia was 759 (95% CI: 589.7–960.8) with variations across areas: 913 (95% CI 696.7–1,176.7; Sumatra), 593 (95% CI 447.2–770.6; Java-Bali), and 842 (95% CI 634.7–1,091.8; other islands). Also, the prevalence of TB varied across diagnostic approaches: 256.5 (sputum AFB), 545 (sputum culture), 752.2 (chest x-ray), and 894.9 (sputum genetic test). Based on sputum AFB, the TB prevalence varied from 216.6 (95% CI 146.5–286.8; Java-Bali), 259.9 (95% CI 184.2–335.6; other islands) to 307.4 (95% CI 208.3–406.5; Sumatra). Based on sputum culture, the TB prevalence ranged from 487.9 (95% CI 433.6–548.6; Java-Bali), 635.9 (95% CI 564.9–715.1; Sumatra), to 2,129.8 (95% CI 1,664.0–2,735.6; other islands). Based on chest x-ray, the TB prevalence varied from 152.1 (95% CI 147.9–156.3; Java-Bali), 159.2 (95% CI 154.1–164.3; Sumatra), to 864 (95% CI 809–921.4; other islands). Based on sputum genetic test, the TB prevalence ranged from 838.7 (95% CI 748.4–900.8; Java-Bali), 875 (95% CI 775.4–934.2; Sumatra), to 941.2 (95% CI 663.6–992.3; other islands). Conclusions The variation of TB prevalence across geographical regions could be confounded by the diagnostic approaches. Trial registration This study was approved by the Institutional Review Board of Chulalongkorn University (IRB No. 684/63).