Background: The incidence of nontuberculous mycobacteria (NTM) infection in Korea is increasing. The aim of this study was to evaluate trends of the recovery rate of Mycobacterium tuberculosis or NTM from positive result either by acid fast bacilli (AFB) staining or culture over a 12-year period in a tertiary hospital in Korea. Methods: We identified all respiratory specimens with positive result from culture regardless of AFB staining results at the Samsung Medical Center (Seoul, South Korea) obtained from January 2000 to December 2011.Results: During the study period, 33,792 isolates from 11,058 patients were positive for mycobacterial culture. The proportion of the NTM isolates from specimens with positive result of mycobacterial culture was increasing from 22.8% (217/951) in 2000 to 69.6% (3,341/4,800) in 2011 (p＜0.001, test for trend). Among the AFB smear-positive specimens, recovery rate of the NTM isolates was also increasing from 6.1% (20/330) in 2000 to 64.3% (1,284/1,997) in 2011 (p＜0.001, test for trend). The most frequently isolated organisms in NTM isolates were Mycobacterium avium complex (n=6,000, 53%) and Mycobacterium abscessus (n=2,864, 25%). Conclusion: We found that the rate of NTM isolation from respiratory specimens has increased over a 12-year period in a tertiary hospital in Korea, and half of the patients with AFB smear-positive respiratory specimens have NTM infection rather than pulmonary tuberculosis.