The most prevalent clinical entity of extra pulmonary tuberculosis is tuberculous lymphadenitis. However, it resembles other granulomatous conditions pathologically and obtaining tissue for microbiological diagnosis is also difficult. Thus it is a challenging task for diagnosis and early initiation of management. Fine needle aspiration cytology and biopsy are the diagnostic methods generally used to obtain the lymph node samples for histopathological and microbiological diagnosis. Mycobacterium culture on Lowenstein-Jensen medium remains the gold standard for definitive diagnosis, but its major limitations is a prolonged turn-around time of 2-4 weeks. The GeneXpert Mtuberculosis/RIF assay is a novel molecular diagnostic method for rapid diagnosis of tuberculosis and rifampicin resistance in clinical specimens. This was a cross sectional analytical study conducted on 67 cases of suspected tubercular lymphadenitis at R.L Jalappa Hospital and Research Centre, Tamaka, Kolar. The study was carried out between December 2017 to June 2019. The samples were collected using excision biopsy and subjected to GeneXpert Mtuberculosis/RIF assay and histopathology. Further, sensitivity, specificity, positive predictive value and negative predictive value was measured and compared with histopathology. The average age of the patients was 37.04 ± 19.27 and majority was males. The lymph nodes were predominantly present in cervical region. Histopathology analysis reveals 46 positive cases of tuberculosis Lymphadenitis and GeneXpert Mtuberculosis/RIF assay detects 42 cases of tuberculosis Lymphadenitis. In the present study, GeneXpert Mtuberculosis/RIF assay had a sensitivity of 82.60% and specificity of 85% when compared to histopathology. Further the PPV and NPV was found to be 92.68% and 68% respectively. GeneXpert Mtuberculosis/RIF showed 2 cases of rifampicin resistance out of 67 cases. In this study, the GeneXpert Mtuberculosis/RIF showed the results in 0.79 days. The present study showed that GeneXpert Mtuberculosis/RIF is a simple and reliable technique for diagnosing tuberculosis Lymphadenitis with high specificity and sensitivity as compared histopathology. Further, the methods elicit rapid diagnosis and also detected rifampicin resistance. It is thus a reliable and useful diagnostic modality in rapid detection of the causative agent and initiation of appropriate category anti-tubercular therapy when necessary. © 2020 The Author(s).