Mycobacterium tuberculosis genotype distribution is different depending on the geographic regions, but the data from Surabaya City in East Java, Indonesia, not reported yet. This study aimed tuberculosis patients in the city for the predominant M. tuberculosis genotypes that can be responsible for tuberculosis in the region. Sputum from 121 tuberculosis patients were collected at Dr. Soetomo General Hospital (Surabaya, Indonesia) in two years starting from September 2016. These samples were tested using the previously developed 16S rRNA sequencing method. A high degree of genetic diversity was observed among isolates based on their 16S rRNA sequences. Further, this study can used for good information in molecular epidemiology, especially in Indonesia.