Background Current microbiologic criteria for nontuberculous pulmonary disease (NTM-PD) require cultures with two separate sputum specimens or one non-sputum specimen. However, data on NTM-PD prognosis following non-sputum culture-based diagnosis are limited. We compared the prognosis of NTM-PD diagnosed using non-sputum and sputum cultures. Methods We analyzed 1020 patients in whom NTM was isolated; they were divided into the sputum NTM isolation (n=170), sputum NTM-PD (n=688), and non-sputum NTM-PD groups (n=162). Initiation of therapy and radiographic aggravation was compared between groups.Results Overall median observation time was 44.3 months (interquartile range, 28.0-70.2). Fewer non-sputum NTM-PD patients initiated treatment than sputum NTM-PD patients (p<0.001). Moreover, fewer non-sputum NTM-PD patients exhibited radiographic aggravation than sputum NTM-PD patients (p=0.001). Both time to radiographic aggravation and initiation of therapy were significantly shorter in the sputum NTM-PD group (log-rank test; p=0.011 and p<0.001, respectively). Sputum NTM-PD patients showed higher initiation of therapy (aHR, 1.38; 95% CI, 1.02-1.87) and risk of radiographic aggravation (aHR, 1.47; 95% CI, 1.02-2.10) than non-sputum NTM-PD patients.Conclusion Prognosis of NTM-PD patients diagnosed with a non-sputum culture was better than that of NTM-PD patients diagnosed with a sputum culture. The disease course may differ depending on the diagnostic method.