Early detection of tuberculosis (TB) is essential for infection control. The geneCube (Toyobo) is a novel fully automated gene analyzer that can amplify target DNAs within 60 min. In this study, we evaluated the ability of the geneCube to directly detect Mycobacterium tuberculosis complex (MTBC) and Mycobacterium avium complex (MAC) in clinical specimens. The results were then compared with those obtained using conventional culture, microscopy, and the Cobas Amplicor assay (Roche). We examined a total of 516 frozen samples from 69 patients who showed culture-positive infection (73 samples; 39 MTBC, 32 MAC, and 2 mixed infections) and from 354 patients who were culture negative (443 samples). Assays using the geneCube had a sensitivity of 85.4% and a specificity of 99.8% for detection of MTBC and a sensitivity of 85.3% and a specificity of 99.8% for detection of MAC. These results are similar to those obtained using the Amplicor system but were obtained much more rapidly (1 h with the geneCube versus 5.5 h with the Amplicor system). The geneCube thus enables a significant shortening of the assay time with no loss of sensitivity or specificity.