Background: Diagnosis of tuberculosis in children is challenging and fine needle aspiration biopsy (FNAB) is used worldwide in the diagnosis of palpable masses including peripheral lymphadenopathy. Recent studies of the use of nucleic acid amplification such as the Xpert MTB/RIF test on FNAB in adult patients have shown considerable promise. Xpert MTB/RIF allows for the rapid diagnosis of Mycobacterium tuberculosis and identification of rifampicin susceptibility. Studies to date have been predominantly performed in adults. This study aims to determine the accuracy of Xpert MTB/RIF for the detection of M. tuberculosis complex in FNAB from children with clinically suspected mycobacterial lymphadenitis. Methods: Prospective hospital-based study of children <13 years referred for FNAB at Tygerberg hospital and Dora Nginza hospital, South Africa, for suspected mycobacterial lymphadenitis. Aspirates were performed and the results of the Xpert MTB/RIF test were compared with liquid (mycobacterial growth indicator tube) culture and cytology. Results: FNABs were collected from 110 children and 38 (35%) cases were excluded. Of the 72 cases included in the study, 32 were positive for M. tuberculosis complex on Xpert MTB/RIF, 36 on cytology and 25 were culture positive for M. tuberculosis complex. Compared with the combined reference standard (cytomorphology suggestive of mycobacterial disease with direct visualization of the organism and/or bacteriological culture), Xpert MTB/RIF identified 32 of 40 cases as positive with a sensitivity and a specificity of 80% and 93.8%, respectively. Conclusions: FNAB and Xpert MTB/RIF enable a rapid diagnosis in pediatric mycobacterial lymphadenitis, expediting appropriate treatment and potentially preventing morbidity and mortality.