The aim of this study was to evaluate the clinical performance of AdvanSure GenoBlot assay using NTM isolates and clinical specimens. A total of 136 NTM isolates and 176 clinical specimens were included in this study. AdvanSure Mycobacteria GenoBlot assay was performed according to the manufacturer's instructions. We compared the results with those of 16S rRNA and rpoB genes sequencing. Of the total 136 isolates, 111 (81.6%) were correctly identified to the species level by the GenoBlot assay. The final concordance rate was 89.7% (122/136) when including 11 GPC results for uncommon NTM. The most common NTM, M. avium, M. fortuitum, M. gordonae, M. intracellulare, M. chelonae, M. abscessus, and M. kansasii, were correctly identified by the GenoBlot assay. For 176 organisms in clinical specimens, 117 were identified to the species level, including a single species for 111 and two species for 6. The final detection and identification rate for clinical specimens was 94.9% and 66.5%, respectively. The AdvanSure GenoBlot assay performs well in identifying most common NTM and would be useful in a clinical laboratory.