<p>Tuberculous lymphadenopathy presents a challenging diagnostic scenario, particularly in regions with high tuberculosis (TB) prevalence. Abdominal TB accounts for 10% of extrapulmonary TB cases. Diagnosing abdominal TB is difficult due to nonspecific clinical, radiological, and endoscopic findings. Blood and skin tests for TB exhibit variable sensitivities and specificities; therefore, histopathological diagnosis and molecular testing of tissue samples may provide greater accuracy than blood-based tests. Obtaining tissue samples under ultrasonographic or CT guidance can be difficult and risky, especially in intra-abdominal and mediastinal regions. Endoscopic ultrasound (EUS) and EUS-guided fine-needle biopsy (EUS-FNB) offer a safe technique for obtaining tissue samples for the diagnosis of abdominal and mediastinal TB. Comparative analyses of the Gene X-pert MTB/RIF assay and PCR techniques demonstrate nuanced diagnostic capabilities. Gene X-pert enables rapid molecular detection with high specificity for rifampicin resistance, whereas PCR facilitates molecular amplification of bacterial DNA. EUS provides the critical advantage of real-time imaging and precise tissue sampling. Several studies have found that EUS-FNB yields significantly higher diagnostic accuracy in complex TB presentations, with detection rates ranging from 78 to 92% across various anatomical sites. This review explores the diagnostic efficacy of molecular techniques, specifically polymerase chain reaction (PCR) and the Gene X-pert MTB/RIF assay, in detecting Mycobacterium tuberculosis in tissue samples obtained through EUS-FNB. Additionally, we evaluated the sensitivity, specificity, and rapid detection capabilities of these molecular methods compared to traditional diagnostic techniques. We further discussed whether molecular techniques such as PCR and Gene X-pert provide a powerful diagnostic strategy for TB lymphadenopathy in EUS-FNB tissue samples, thereby overcoming the limitations of conventional diagnostic methods.</p>