In India, tuberculosis remains a major public health problem and Tuberculous lymphadenitis remains a diagnostic challenge. This study aimed to isolate M. tuberculosis from lymphadenitis employing LJ and MGIT culture. A total of 49 single clinical specimens collected from patients with clinical suspicion of Tuberculous lymphadenitis were included. Ziehl-Neelsen microscopy and culture was performed and growth of M. tuberculosis was identified using standard tests. Drug susceptibility testing was carried out in MGIT and the isolated M. tuberculosis strains causing lymphadenitis were genotyped by spoligotyping. Among 49 samples processed, 28.6% were positive by ZN, 20.4% on LJ and 26.7% in MGIT. LJ and MGIT together yield 32.7% positivity. The mean and median turnaround time for LJ and MGIT culture was 31.8 and 14.5 days respectively. Conventional identification tests identified 15 M. tuberculosis strains and one M. bovis BCG. Drug susceptibility revealed 50% isolates were resistant to one or more drugs, but not MDR-TB. Spoligotyping revealed 47% of the strains causing lymphadenitis were profiles described as orphans. To conclude, inclusion of both LJ and MGIT increases percentage positivity. The short Turn Around Time (TAT) of MGIT helps early reporting of drug susceptibility thus avoiding empirical treatment, in areas where molecular techniques are not feasible. Orphan spoligotype is associated with lymphadenitis.