The aim of this study was to characterize Mycobacterium tuberculosis isolates circulating in the Amhara Region of Ethiopia. Sputum samples were collected from new pulmonary tuberculosis (TB) patients in the Region. Genotyping of mycobacterial DNA was performed by spoligotyping and isolates were assigned to families using the SpolDB4 and the model-based program ‘Spotclust’. A high level of diversity was found among the 237 isolates. Sixty-five different spoligopatterns were obtained. The T (30.8%), Central Asian (CAS; 21.1%) and U (17.7%) families were the predominant isolates comprising 69.6% of the total strains. Eighty-five per cent of the U lineage belonged to Spoligo-International-Type (SIT) 910 and SIT 1729. Only a few of these strains are included in SpolDB4 to date. Of the total strains, 41 (17.3%) were unique and have not been described in SpolDB4 to date. This study indicated that the TB epidemic in Amhara Region, Ethiopia, is characterized by the circulation of numerous M. tuberculosis strain families. The high proportion of SIT 910 and SIT 1729 strains may indicate an increase in the importance of these lineages in the transmission of TB in the study region.