Introduction: Tuberculosis (TB) is one of the major killers among infectious diseases in thernworld. Each year an estimated 8.9 to 9.9 million incident cases and approximately 1.55 torn2.32 million deaths due to TB occur worldwide. Ethiopia ranks 7thrnin the list of worldâ€™s 22rnhigh burden countries for TB with incidence estimated at 379 persons per 100,000 persons forrnall forms of TB. Based on preliminary assessment about the disease and the research gap inrnthe study setting, a cross-sectional study was carried out from November 2010 to June 2011rnon 99 smear positive pulmonary and 98 smear negative extrapulmonary TB patients at DebrernBirhan Referral Hospital.rnObjective: Molecular characterization of Mycobacterium strains implicated in tuberculosis.rnMaterials and methods: Structured questionnaire, acid fast bacilli smear staining, culture,rndeletion typing and spoligotyping were used in the study.rnResults: The proportional distribution of TB disease and isolates were not varied substantiallyrn(p>0.05) either with age, sex, residence area, occupation, previous contact with TB patients,rnprevious treatment with antibiotics or antimycobacterial drugs, habit of raw animal productrnconsumption or with close contact with livestock for both pulmonary and extrapulmonary TBrncombined. Out of 99 sputum and 98 fine needle aspirate samples, 80.80% (80/99) and 36.73%rn(36/98) of them were culture positive respectively. Speciation of isolates using region ofrndifference-9 (RD9) deletion typing showed that 90.00% (72/80) of the isolates from sputumrnand 88.89% (32/36) of the isolates from fine needle aspirate were M. tuberculosis, while onlyrn1.25% (1/80) and 2.78% (1/36) were M.bovis respectively. Further characterization of 97 M.rntuberculosis isolates to the strain level using spoligotyping resulted in the identification of 25rnclusters that constituted 80.41% (78/97) of the isolates tested out of which 17 clusters werernnew to Ethiopia. The most dominant spoligotypes were spoligo international typing numberrn(ST) 149, 53 and 37 that accounted 32.99% (32/97) of the total spoligotypes identified.rnComparison of our spoligotypes with international spoligotype database, SpolDB4, showedrn19 new spoligotypes which were clustered into nine clusters and have never been reportedrnfrom elsewhere in the world. Analysis of non-clustered (new) spoligotypes and their sourcernindicated that 35.71% (10/28) of the isolates from extrapulmonary sources were unique,rncompared with 13.04% (9/69) of the pulmonary isolates. Classifying strains on the bases ofrnphylogeny of M. tuberculosis using SPOTCLUST software revealed that they belonged tornEuro-American, East-African Indian and Mycobacterium africanum lineages. The mostrnviiirnprevalent lineage in the current investigation was Euro-American constituting 69.07% (67/97)rnof the strains analyzed.rnConclusion: This research has shown the presence of several clusters and new strains of M.rntuberculosis circulating both in pulmonary and extrapulmonary TB patients in Ethiopia. Asrnmapping the population structure of M. tuberculosis is vital to understand the transmissionrnand disease dynamics TB and set appropriate control measure, further similar studies arernrecommended.rnKey words:rnRD9 Typing, Spoligotyping, Mycobacterium family, Mycobacterium lineage, Tuberculosis,rnDebre Birhan Referral Hospital