The Xpert MTB/RIF (Xpert) test is a novel automated molecular diagnostic recently endorsed by the World Health Organization for rapid diagnosis of tuberculosis (TB). Nevertheless, performance related data from high TB prevalence regions to investigate clinically suspected TB lymphadenitis are limited. To evaluate the performance of Xpert test for direct detection of the Mycobacterium tuberculosis complex (MTBC) and rifampicin (RIF) resistance in lymph node aspirates, a cross-sectional study was conducted at four main hospitals in northern Ethiopia. Culture served as a reference standard for growth of MTBC and phenotypic and MTBDRplus drug susceptibility testing for detecting RIF resistance. Two-hundred-thirty-one fine needle aspirate (FNAs) specimens were processed simultaneously for smear, culture, and Xpert test. When compared to culture, the Xpert test correctly identified 29 out of 32 culture positive cases, 5 out of 11 contaminated cases, and 56 out of 188 culture negative cases. The overall sensitivity of the test was 93.5% [95% CI, 78.3–98.9%] and specificity 69.2% [95% CI, 66.4–70.0%]. The Xpert test identified the rpoB mutations associated with RIF resistance concordant with GenoType MTBDRplus and phenotypic drug susceptibility testing. In conclusion, the Xpert assay was found to perform well in detecting MTBC and RIF resistance in TB lymphadenitis patients. Furthermore, the test is simple and suitable to use in remote and rural areas for the diagnosis of TB lymphadenitis directly from FNAs in Ethiopia where TB/MDR-TB is rampant.