Background: The diagnosis of tuberculous lymphadenitis (TBLN) ranges from therapeutic diagnosis to open biopsy with tissue culture. The open biopsies are accepted as the gold standard to diagnose TBLN, but it requires skin incision that leaves unwanted scars. Objective: Test the sensitivity and specificity of fine needle aspiration (FNA) using tissue culture in mycobacteria growth indicator tube (MGIT) and tissue polymerase chain reaction (PCR) for comparison with open biopsy using tissue culture. Subject and methods: Forty patients with clinically suspected cervical tuberculous lymphadenitis were recruited at King Chulalongkorn Memorial Hospital. The patients underwent FNA followed by open biopsies either excisional or incisional. Specimens from FNA were collected for tissue culture in MGIT and for tissue PCR. The specimens from open biopsies were divided into two portions for tissue culture in MGIT (the gold standard) and for hispathology. Results: FNA for tissue culture in MGIT had a moderate sensitivity (65%) but high specificity (83%) (73% positive and 76% negative predictive value). FNA for tissue PCR had a moderate sensitivity (53%) but very high specificity (96%) (90% positive and 73% negative predictive values). Combination of either FNA for tissue culture or FNA tissue PCR revealed an increase in sensitivity and specificity to 83.6% and 80.0%, respectively. However, a combination of both FNA for tissue culture and FNA tissue PCR revealed a decrease in sensitivity (34.5%) but a highly increase in specificity (99.0%). Conclusion: Either the FNA using tissue culture in MGIT or tissue PCR had a moderate sensitivity but high specificity. FNA using tissue culture or FNA tissue PCR may be used as an alternative test for diagnosis TBLN. The techniques may replace the open biopsies because of its effectiveness and low complication rate.