Molecular diagnostic methods are of potential value in identifying tuberculosis (TB) and drug resistance where tissue specimens have been submitted for histology but not for microbiological culture. All paraffin-embedded tissue (PET) specimens (n=60) referred to a single national centre over a 42 month study period were analysed using the INNO-LiPA Rif.TB assay; 29/60 patients had been reported to the UK Enhanced Tuberculosis Surveillance database with a diagnosis of TB based on clinical, radiological and histological evidence. Mycobacterium tuberculosis (MTB) DNA was detectable in 5 out of 29 reported TB cases (17.2 %); 12 out of 29 of the reported TB cases had a positive MTB culture from a secondary clinical specimen and MTB DNA was detectable in 2 of the 12 (16.7 %) laboratory-confirmed TB cases. Referring clinicians should be aware of the limitations of this assay on PETs, and should request molecular testing only in patients with a high clinical probability of TB and when acid-fast bacilli are seen in tissue specimens.