Tuberculosis kills over 1.7 million people worldwide every year and nearly 40% of patients with active tuberculosis remain undiagnosed because of the poor sensitivity of the current, century old diagnostic method: sputum microscopy. Sputum microscopy is not able to easily detect paediatric, extrapulmonary, or HIV-associated tuberculosis, which are now important causes of morbidity and mortality in developing countries. Newer diagnostic methods for tuberculosis remain less sensitive than sputum microscopy. Alternative strategies to diagnose tuberculosis by use of nucleic acid amplification methods to detect fragments of mycobacterial DNA in urine have been developed over the past decade with varying sensitivities and specificities. Methods using quantitative PCR on urine samples to detect transrenal mycobacterial DNA are under development. The detection of transrenal DNA makes it possible to assay the total body burden of mycobacterial infection in any age group and in extrapulmonary tuberculosis with urine samples, which can be collected non-invasively. This Review discusses the developments and application of nucleic acid amplification of mycobacterial transrenal DNA for improved tuberculosis diagnostics.