Background: There is a global increase in isolation of nontuberculous mycobacteria (NTM). The aim of the study was to analyze longitudinal trends of NTM identification and pattern of antimicrobial susceptibility testing.Methods: NTM recovery rates, distribution of NTM species identification, and antimicrobial susceptibility pattern of NTM at Pusan National University Yangsan Hospital between January 2016 and December 2020 were retrospectively analyzed.Results: A total of 52,456 specimens from 21,264 patients were submitted for mycobacterial culture, of which 2,521 from 1,410 patients were NTM positive over five years (January 2016 to December 2020). NTM isolation showed an increasing trend from 2016 to 2020 (p<0.001, test for trend) mainly caused by <i>Mycobacterium avium</i> complex. The vast majority of <i>M. avium</i> complex were susceptible to key agents clarithromycin and amikacin. For <i>Mycobacterium kansasii</i>, resistance to rifampin and clarithromycin is rare. Amikacin was the most effective drug against <i>Mycobacterium abscessus</i> subspecies <i>abscessus</i> and <i>Mycobacterium</i> subspecies <i>massiliense</i>. Most of <i>M.</i> subspecies <i>massiliense</i> were susceptible to clarithromycin, while the majority of <i>M. abscessus</i> subspecies abscessus were resistant to clarithromycin (p<0.001).Conclusion: There was an increasing trend of NTM isolation in our hospital. Resistance to key drugs was uncommon for most NTM species except for <i>M. abscessus</i> subspecies <i>abscessus</i> against clarithromycin.