Tuberculosis (TB), a bacterial infectious disease, continues to be a public health concern in many developing countries. However, lack of data concerning the public health burden and potential risk factors for the disease hampers control programs in target areas. Therefore, the aims of present study were to determine the prevalence of TB and genetic diversity of M. tuberculosis isolates from individuals visiting health facilities in South Gondar Zone, northwest Ethiopia. A cross-sectonal study was conducted between March 2015 and April 2017. Bacteriological examination, region of difference (RD) 9 based polymerase chain reaction (PCR) and spoligotyping were used. The overall prevalence of all smear positive TB was 6.3% (186/2953). Extra pulmonary TB (EPTB) was clinically characterized in about 62.4% (116/186) TB-positive cases. Some demographic characteristics, such as patients' origin (districts where patients were recruited) patients' origin (chi-square (χ2) value; 62.8,p<0.001) were found to be significantly associated risk factors for the occurrence of TB in the study area. All the mycobacterial isolates were found to be M. tuberculosis. Among the 35 different spoligotype patterns identified, 22 patterns were shared types.The three dominantly identified families were T, CAS and Manu, each consisting of 46.9%, 24.0% and 10.4% of the isolates, respectively. The present study revealed that TB continues to be a public health problem in South Gondar Zone which suggests a need of implementing effective disease control strategies.