The role of GeneXpert MTB/RIF (GX) in tuberculous (TB) mastitis is not defined. The aim of this study was to evaluate the role of GX in the diagnostic evaluation of TB mastitis. Patients presenting with signs and symptoms of breast abscess, previously drained breast abscess that did not heal, and recurrent breast abscesses were included in the study. GX (cartridge-based nucleic acid amplification test), FNAC, abscess wall scrapings for histopathology, pus for culture and sensitivity, chest X-ray, serum adenosine deaminase (ADA), and ESR levels were performed in all patients. Female patients (n = 228) were enrolled in the study between August 2013 and December 2017. A total of 26 (11.4%) patients were diagnosed with TB mastitis using multiple clinical/biochemical/histopathological parameters. GX was positive for Mycobacterium TB (MTB) in 22 patients whereas FNAC and combined ADA+ESR (ADA > 23.5, ESR > 25) were suggestive of TB mastitis in 6 and 18 patients respectively. The sensitivity, specificity, positive predictive value, and negative predictive value for GX test, FNAC, and combined ADA+ESR were 83.3%,