Tuberculosis is a chronic debilitating infectious disease causing a severe challenge to public health, especially in developing countries. The aim of this study was to examine genetic diversity in Mycobacterium tuberculosis strains circulating in the Balochistan region of Pakistan.One hundred isolates collected from patients visiting the Fatima Jinnah TB Hospital in Quetta were subjected to genotype analysis by spoligotyping.Three main genotypes were identified: Central Asian Strain 1 (CAS1) (n = 89), East African Indian (EAI) strain (n = 7) and Latin American Mediterranean (LAM) strain (n = 3). The CAS1 clade (ST 26) had high genetic diversity represented by seven different spoligopatterns, of which one had major predominace (n = 75).This is the first insight into the genotype of M. tuberculosis strains in the Balochistan region that might serve as a base line study for control of tuberculosis in the community.