A cross sectional study was conducted on 2231 small ruminants in four districts of the Afar Pastoral Region of Ethiopia to investigate the epidemiology of tuberculosis in goats and sheep using comparative intradermal tuberculin skin test, postmortem examination, mycobacteriological culture and molecular typing methods. The overall animal prevalence of TB in small ruminants was 0.5% (95% CI: 0.2%-0.7%) at ≥4 mm and 3.8% (95% CI: 3%-4.7%) at cutoff ≥2 mm. The herd prevalence was 20% (95% CI: 12-28%) and 47% (95% CI: 37-56%) at ≥4 mm and ≥2 mm cut-off points, respectively. The overall animal prevalence of Mycobacterium avium complex infection was 2.8% (95% CI: 2.1-3.5%) and 6.8% (95% CI: 5.8-7.9%) at ≥4 mm and ≥2 mm cut-off points, respectively. Mycobacteriological culture and molecular characterization of isolates from tissue lesions of tuberculin reactor goats resulted in isolation of Mycobacterium tuberculosis (SIT149) and non-tuberculosis mycobacteria as causative agents of tuberculosis and tuberculosis-like diseases in goats, respectively. The isolation of Mycobacterium tuberculosis in goat suggests a potential transmission of the causative agent from human and warrants further investigation in the role of small ruminants in epidemiology of human tuberculosis in the region.