In recent years, a rise in non-tuberculosis mycobacteria pulmonary disease (NTM-PD) has been reported in several countries. However, data for high-burden tuberculosis settings, including South Africa, is currently limited. In this study, we conducted a retrospective analysis of routine diagnostic data obtained from one diagnostic laboratory in South Africa between 2015 and 2019. During this period, samples from 275 individuals with suspected mycobacterial infection were tested using the GenoType Mycobacterium CM (Common mycobacteria) or AS (Additional species) line probe assay (LPA) (Brucker-Hain Life science, Nehren, Germany), yielding an NTM-positive result for 163 of these individuals. Interestingly, the positivity rate in respiratory samples declined from 93 % in 2015 to 79 % in 2019. Just over half of the positive samples were of respiratory origin, and the most common species identified in respiratory samples was Mycobacterium intracellulare/Mycobacteium avium complex (28.9 %), followed by M. avium (17.4 %). Where the mycobacterial species was not identified by the LPA, a higher proportion of the subsequent cultures were negative, suggestive of colonisation rather than infection. More than half of patients with a positive NTM-LPA were HIV positive (55.9 %), and this association declined slightly during the study period (62.5 %-50 %).