Background: Diagnosis of smear negative pulmonary tuberculosis (SNPTB) is challenging, especially in patients with HIV. The Botswana National Tuberculosis Program (BNTP) guidelines give guidance in diagnosing and treating SNPTB. Patients with chronic cough should be screened for TB by 3 sputum smear investigations. If negative, a chest x-ray (CXR) should be performed. If negative for TB, antimicrobial treatment for other infections should be started. We investigated the clinicians' use of the guidelines in clinical practice. Methods: Data regarding the medical history (coughing period), requested and conducted investigations concerning tuberculosis diagnosis (sputum smear or culture or CXR) or alternative diagnoses (sputum microscopy or blood or sputum culture for diagnosis of other organisms), in SNPTB HIV-positive patients (outpatients and admitted patients) from 2006-2009 in a district hospital in Botswana were extracted from all available hospital medical records. Additionally, a survey was done in all doctors diagnosing SNPTB in this hospital using a self-administered questionnaire with questions regarding the application of the BNTP guidelines in practice. Descriptive analyses of collected data were performed to test the compliance to the guidelines. Results: Data from medical records showed that in 47.0% (132/281) of patients, TB treatment was started without microbiological results from sputum smears. Other methods to rule out or confirm PTB were used in 2.1% (6/281); and 99.6% (280/281) of SNPTB patients had received a CXR. The survey in 7 clinicians found that all always used CXR, and all clinicians requested three sputum results only sometimes. Six out of 7 clinicians started antibiotics before starting TB treatment. Reasons clinicians gave for difficulties in following the guidelines included inability of patients to produce sputum; and laboratory delays in releasing sputum results. Conclusion: Between 2006 and 2009 a high proportion of SNPTB diagnoses in a district hospital in Botswana were not supported by laboratory investigation.