Background: Recent increases in the incidence of Nontuberculous mycobacteria (NTM) infections are of concern, and it is important to identify NTM accurately for efficient treatment. NTM identification based on line probe assay has been usually performed. However, it is hard to be objective because of band reading method visually. This study aimed to evaluate newly developed AdvanSure Mycobacteria GenoBlot Assay (LG Life Sciences, Korea) compared to GenoType Mycobacterium CM/AS assay (HAIN Life science) using NTM difficult to identify. Methods: A total of 71 specimens collected during 22 months from January, 2015 to October, 2016 were used in this study. In cases of the culture- and the acid-fast stain-positive, DNA was extracted and AdvanSure TB/NTM real-time PCR kit (LG Life Science) was used for the selection of NTM. NTM identification was performed using both Genotype mycobacterium CM/AS assay (HAIN method) and AdvanSure Mycobacteria GenoBlot Assay (LG-MGA method). The results by HAIN method were read visually and those by LG-MGA method were analyzed by the automatic analyzer, respectively. The identification results were compared with those of ITS region sequencing. The ID results were compared to those of sequence-based ID and assigned to one of four categories: 1) Correct ID (identical to sequence-based ID), 2) Incorrect ID (either one wrong species was identified, or two or three incorrect species were proposed), 3) Incomplete ID (two or three species were proposed and one was correct) Results: A total of 71 cases included 22 cases of M. intracellulare, 12cases of M. abscessus, 7 cases of M. avium, 4 cases of M. lentiflavum, 3 cases of M. chelonae, 3 cases of M. gordonae, 2 cases of M. kumamotonense, 2 cases of M. terrae, 2 cases of M. ulcerans and 14 cases of the other species. The correct ID rates were 52.1% by HAIN method and 60.5% by LG-MGA method, the incomplete ID rates were 12.7% and 18.3% and the incorrect ID rates were 35.2% and 21.1%, respectively. In especially, the correct ID rates of M. intracellulare and M. abscessus were significantly higher by LG-MGA method than by HAIN method (LG-MGA vs. HAIN, 90.9% vs. 59.1% for M. intracellulare; 83.3% vs. 66.7% for M. abscessus, p-value <0.05, in both). When we compared HAIN method and LG-MGA method, 42 cases (59.2%) were concordant, and 29 cases (40.8%) were discordant. Agreement were varied according to the species as follows: M. intracellulare 72.7% (16/22), M. abscessus 66.7% (8/12), M. avium 85.7% (6/7), M. lentiflavum 25.0% (1/4), M. chelonae 66.7% (2/3), M. gordonae 100% (3/3). Of 29 discordant cases, 12 cases were identical with the results by LG-MGA method and 5 cases were identical with those by HAIN method, respectively. Conclusion: The current study showed that AdvanSure Mycobacteria GenoBlot Assay could yield more correct identification than GenoType Mycobacterium CM/AS assay in case of M. intracellulare and M. abscessus. It might be useful in clinical laboratories because of the advantage of obtaining more objective results.