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Objectives: The primary objective was to determine the association between histopathological and microbiological findings in patients clinically suspected to have tuberculous lymphadenitis. A secondary objective was to assess the diagnostic utility of GeneXpert in lymph node specimens. Method: This was a single-centre prospective cohort study, performed in the Infectious Disease Clinic at The Indus Hospital. Three hundred and forty-one adult patients with chronically enlarged, accessible lymph nodes were enrolled after obtaining verbal consent, between February 2013 and April 2016. Tissue specimens were processed for histopathology, acid-fast bacillus (AFB) microscopy, AFB culture, and GeneXpert. Based on these results, anti-tuberculosis therapy (ATT) was prescribed. Clinical characteristics and treatment outcomes were recorded. Results: There were 297 evaluable patients; 74.4% were diagnosed with tuberculous lymphadenitis (TBLA), 6.7% with a malignancy, and 12.8% with reactive nodes. TBLA was diagnosed on suggestive histopathology in 89.6% of cases, followed by GeneXpert (32.6%), mycobacterial culture (26.6%), and AFB smear positivity (12.5%). The sensitivity of GeneXpert was 65.7% when assessed against AFB culture. Drug resistance was displayed by 8.2% of GeneXpert-positive cases and 11.7% of culture-positive cases. The majority of TBLA patients (88.7%) responded favorably to ATT. Conclusions: In light of laboratory evidence, a quarter of patients suspected of TBLA had an alternative diagnosis, highlighting its importance in avoiding over-treatment and diagnostic delays in malignancy. Although sensitivity is poor, the demonstration of drug resistance by both GeneXpert and AFB culture represents a useful tool to guide treatment. Keywords: Chronic granulomatous inflammation, Mycobacterial culture, GeneXpert, Malignancy, Reactive nodes