In this study, we aimed to assess the performance of Xpert in fresh tissue and formaldehyde-fixed and paraffin embedded (FFPE) specimens from suspected lymphatic tuberculosis for the diagnosis of Mycobacterium tuberculosis (MTB). A total of 52 suspected lymphatic tuberculosis (TB) samples and 10 non-tuberculous lymph nodes samples were collected from outpatients. Using the comprehensive diagnostic criteria as the gold standard, the specificity in fresh and FFPE samples was 100% and the sensitivity was 82.7% and 67.3%, respectively. The majority of fresh tissue specimens had medium and low MTB content, while the low and very low MTB content were noted in 42.9% and 54.3% of FFPE tissue specimens, respectively. There were statistical differences in the MTB content between the two specimen groups detected by Xpert. Three rifampicin-resistant cases in FFPE samples were noted as rifampicin-susceptible in fresh tissue samples. Notably, all three cases with contradictory results of rpoB gene mutation test in fresh and FFPE samples had very low MTB content in FFPE samples. Fresh tissue specimens are more likely to yield Xpert results with high greater MTB content than FFPE specimens from lymphatic TB. The false detection of rpoB mutants is associated with the low bacterial content in the specimens.