Multidrug drug-resistant tuberculosis (MDR-TB) is a major health problem and seriously threatens TB control globally. Ethiopia is among the 30th highest TB burden countries. MDR-TB is a result of unsuccessful TB control programs and associated with mutations in drug target genes. Another challenge of TB is that about one-third of TB/HIV co-infected patients fail to produce a sputum specimen for diagnosis. Understanding of the pattern of resistance and genotyping of strains, and identify alternative specimen for diagnosis are important for TB/MDR-TB control. Thus, the aim of this study was to determine the pattern of mutations associated with resistance, genotype, and performance of GeneXpert MTB/RIF using urine specimens. A cross-sectional study was conducted in Addis Ababa from June 2015 to December 2016. Sputum specimens and socio-demographic data were collected from 358 MDR-TB suspected cases, and 150 urine specimens from sputum scarce cases and cases capable of giving sputum suspected of pulmonary TB. Specimens were analyzed using Ziehl-Neelsen, GeneXpert MTB/RIF assay, and cultures. All culture-positive specimens were analyzed for drug susceptibility tests. Sequence analysis for rpoB, katG, fabG1-inhA, embB, pncA, rrs and rpsL genes, and genotyping was performed using 24 MIRU-VNTR and spoligotype. Data were analyzed using SPSS version 23. rnOf 358 MDR-TB suspected cases, 226 were culture positive for Mycobacterium tuberculosis, of these, 162 (71.7%) had been previously treated for tuberculosis, while 128 (56.6%) were TB/HIV co-infected. An analysis of drug resistance indicated that 110 (48.7%) strains were resistant to isoniazid, 94 (41.6%) to streptomycin, 89 (39.4%) to rifampicin, 72 (31.9%) to ethambutol, and 70 (30.9%) to pyrazinamide. Among the 226 study participants, 89 (39.4%) were determined to be MDR-TB with TB/HIV co-infection, cigarette smoking, alcohol drinking, and admissions and/or visits to hospitals significantly (p