Validation of the MycoAlign assay, a newly developed Mycobacterium spp. identification system based on internal transcribed spacer-1 sequencing, was performed using 50 acid-fast bacilli (AFB)-positive clinical laboratory specimens. Forty-three (86%) diagnostic-level results were obtained, including 38 Mycobacterium spp. and 5 other AFB-positive genera. Three isolates (6%) had suboptimal identity scores with high probability (81-87% identity score). Four (8%) mixed-pattern results were obtained. Forty-five (90%) observations were concordant with the species identification by standard methods, including all controls.