The United Kingdom Health Protection Agency Mycobacterium Reference Unit offers a national Fastrack molecular service for detecting Mycobacterium tuberculosis complex (MTBC) and rifampin resistance by using the INNO-LiPA Rif.TB assay. We analyzed the service in a routine, nontrial context of 1,997 primary clinical specimens, including 658 nonrespiratory specimens. The overall adjusted concordance, sensitivity, specificity, positive predictive value, and negative predictive value for detecting MTBC were 91.2%, 85.2%, 96.2%, 95.7%, and 86.7%, respectively (unadjusted, 86.7%, 85.2%, 88.2%, 86.9%, and 86.7%), when false-positive samples from patients (n = 83) with a known microbiologic diagnosis of MTBC or patients receiving current or recent antituberculous treatment were excluded. The parameters for detecting rifampin resistance were 99.1%, 95.0%, 99.6%, 92.7%, and 99.7%, respectively. The assay enabled earlier diagnosis of MTBC and rifampin resistance (15.2 days) compared with culture-based techniques (30.7 days).