ABSTRACT BACKGROUND: Multi-drug Resistant Tuberculosis (MDR-TB) is found to be a major public health problem both in developed and developing countries. Ethiopia is one of the 30 high MDR-TB burden countries in the world. Although several studies were done to identify the determinants of MDR-TB, the reported findings are heterogeneous across the world.METHODS: Unmatched case-control study was conducted at Debre Markose Referral Hospital, Debre Birhan Referral Hospital, and Boru Media District Hospital in Amhara Region, Northern Ethiopia, from March 01/2019- April 30/2019. Cases were all tuberculosis patients with culture or line probe assay confirmed mycobacterium tuberculosis resistant to at least both Isoniazid and Rifampicin and registered on second-line TB treatment. Controls were all patients with Bacteriological (molecular) proven drug-susceptible TB strains and whose recent smears result were turned to negative and registered as cured from January 01/2014 – December 31/2018. A pre-tested checklist was used to collect the data.RESULT: Of the total reviewed documents (393), 98 cases and 295 controls were involved in this study.  And, 54(55.1%) among cases and 162(54.9%) among controls were males. sixty nine(70.4%) among cases and 163(55.3) among controls were within the age group of 26-45 years. In the multivariable logistic regression analysis, age between 26-45years old (AOR=3.35; 95% CI: 1.15, 9.77), previous history of TB treatment (AOR= 14.2; 95%CI: 7.8, 25.3) and being HIV positive (AOR=4.4; 95% CI: 1.8, 10.4) were significantly associated with MDR-TB.CONCLUSION: Age between 26-45 years old, previously treated cases and TB/HIV co-infection were identified as the determinants of MDR-TB. Special attention should be given to age between 26-45 years old, previous history of TB treatment and TB/HIV co-infection to prevent and control MDR-TB in the local context.KEYWORDS: Ethiopia: multi drug resistant tuberculosis: determinants of microbial resistance