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Objective: To study the sensitivity and specificity of line probe assay (LPA) and GeneXpert compared with drug susceptibility test (DST). Methods: A cross-sectional study was conducted for 126 TB patients in Sudan. Mycobacterium tuberculosis was identified by drug susceptibility test. The smear-positive sputum specimens were screened for rapid detection of resistance to rifampicin (RIF) and isoniazid (INH) by molecular LPA and GeneXpert assay. Results: 67.5% of patients were male, 19.8% were new cases, 57.1% were previously treated TB patients, and 64.3% of the affected patients were in the age group of 16–30 years. About 32.5% of samples were MDR by both DST and LPA, 66.7% were sensitive by both DST and LPA, and 0.79% were MDR by DST and sensitive by LPA. However, 36.51% of samples were MDR by both DST and GeneXpert, and 63.49% were sensitive by both DST and GeneXpert. There was no significant difference between DST and LPA technique (P = 0.50). The sensitivity, specificity, positive predictive value and negative predictive value of LPA were 97.6%, 100%, 97.6%, and 100%, respectively; while for GeneXpert, they were 100%, 100%, 100% and 100%, respectively. The RIF resistance was associated with mutation in the region of rpoB 530-533, mostly S531L mutation, and the most INH resistant samples (98.58%) were linked with KatG gene and codon 315 (S315T1). Conclusions: The molecular assays are the best screening tools for MDR TB, which may exceed TB diagnoses.