Background: Globally, tuberculosis (TB) has affected human beings for thousands of years andrnthe emergence of drug-resistant tuberculosis has been a major obstacle to control the disease,rnespecially in developing countries including Ethiopia. rnObjective: The study investigated the molecular epidemiology and drug resistance pattern of M.rntuberculosis circulating in the Amhara, Gambella and Benshangul-Gumuz regions of Ethiopia.rnMethods: A cross-sectional study was conducted using 128 MTBC isolates that were obtainedrnfrom presumptive TB/MDR-TB patients. A total of 127 recovered isolates were used to investigaterndrug resistance patterns with the indirect proportion method using the MGIT 960 system and therngenotypic method at the Ethiopian Public Health Institute. Further, identification of mycobacterialrnstrain types and mycobacterial lineages was done using spoligotyping. Data were managed usingrnthe Epi-info, SPSS version 25, SITVIT2, MIRU-VNTRplus and TBInsight databases. rnResults: Of the 127 recovered mycobacterial isolates, 100 (78.7%) were sensitive to the four firstlinernanti-TBrndrugs,rnbutrn27rn(21.3%)rnwerernresistantrntornatleastrnonernanti-TBrndrug,rn20rn(15.7%)rnrnwerernpoly-resistant, 17 (13.4%) were multi-drug resistant and 2 (10.5%) were Pre-XDR. The highestrngene mutations associated with INH and RIF resistance were observed in the katG MUT1 genern(S315T1) [20 (76.9%)] and rpoB gene (S531L) [10 (52.6%)], respectively. Further, the two preXDRrnrncases showed mutations in the gyrA gene. Among the 127 spoligotyped isolates, 119rngenerated 43 different spoligotyping patterns; 87 (73.1%) generated 26 distinct spoligotypernpatterns; and 31 (26.0%) generated 17 different spoligotype patterns. The predominantly identifiedrnstrains, family, lineages and sublineages were the SIT149 (19, 15.9%), T family (54, 45.4%), EuroAmericanrn(72,rn60.5%)rnandrnCAS1-Delhirn(24,rn20.2%),rnrespectively.rnAdditionally,rnMycobacteriumrnrnAfricanumrn(12,rn10.1%)rnwasrnidentified.rnFurthermore,rndrugrnresistancernwasrnsignificantlyrnassociatedrnrnwithrnpreviousrnTBrnhistoryrn(Xrn2rn=46.59; p