Tuberculosis remains the leading cause of death from an infectious disease worldwide. Early and accurate diagnosis and detection of drug-sensitive and drug-resistant tuberculosis is essential for achieving global tuberculosis control. Despite the introduction of the Xpert MTB/RIF assay as the first-line rapid tuberculosis diagnostic test, the gap between global estimates of incidence and new case notifications is 4·1 million people. More accurate, rapid, and cost-effective screening tests are needed to improve case detection. Diagnosis of extrapulmonary tuberculosis and tuberculosis in children, people living with HIV, and pregnant women remains particularly problematic. The diagnostic molecular technology landscape has continued to expand, including the development of tests for resistance to several antituberculosis drugs. Biomarkers are urgently needed to indicate progression from latent infection to clinical disease, to predict risk of reactivation after cure, and to provide accurate endpoints for drug and vaccine trials. Sophisticated bioinformatic computational tools and systems biology approaches are being applied to the discovery and validation of biomarkers, with substantial progress taking place. New data have been generated from the study of T-cell responses and T-cell function, serological studies, flow cytometric-based assays, and protein and gene expression studies. Alternative diagnostic strategies under investigation as potential screening and triaging tools include non-sputum-based detection with breath-based tests and automated digital radiography. We review developments and key achievements in the search for new tuberculosis diagnostics and biomarkers. We highlight gaps and challenges in evaluation and rollout of new diagnostics and biomarkers, and prioritise areas needing further investment, including impact assessment and cost–benefit studies.