Tuberculosis is one of the oldest diseases known to affect humans and is caused by Mycobacterium tuberculosis. Lymph nodes are the most common site of extrapulmonary involvement. Tuberculous lymphadenitis can be presumptively diagnosed morphologically on fine-needle aspiration biopsy of lymph nodes. Additional tests like Ziehl-Neelsen (ZN) stain for detection of acid fast bacilli, fluorescence, and molecular markers are often employed for confirmation of diagnosis. However, culture is essential for obtaining a definitive diagnosis. Unfortunately, culture is time consuming and expensive. Newer investigative methods are required. The efficacy of autofluorescence in the diagnosis of tuberculous lymphadenitis was evaluated for this purpose. Fine-needle aspiration biopsies were collected from patients with a clinical diagnosis of tuberculous lymphadenitis. Cytomorphological examination, ZN staining, autofluorescence, and culture were performed on all specimens. ZN staining was positive in 37.5% (30/80) of cases, while autofluorescence was positive in 57.5% (46/80) of patients and was found to have 81.8% specificity, 95% sensitivity, and a positive predictive value of 82.6%. This technique allowed rapid and early diagnosis of tuberculous lymphadenitis in a number of patients, permitting early institution of appropriate therapy. Diagn. Cytopathol. 2013;41:330–334. © 2012 Wiley Periodicals, Inc.