Conventional methods for identification of nontuberculous mycobacteria (NTM) are often inexact and time consuming. Sequencing of bacterial 16S rDNA is accurate, rapid and effective. We have retrospectively evaluated the discriminative power of pyrosequencing of a short hypervariable 16S rDNA fragment as a simple and rapid tool for NTM characterization. A series of 312 clinical NTM isolates, excluding the M. avium/intracellulare complex, was investigated. When species could not be resolved by sequencing alone, growth rate and pigment production were also examined. 54% (170/312) of the isolates were unambiguously identified by both methods. An additional 14% (45/312) were directly identified to species by conventional 16S rDNA sequencing but needed complementary phenotypic analysis when examined by pyrosequencing. The remaining 31% (97/312) needed additional phenotypic analysis for both sequencing methods. We consider the pyrosequencing procedure to be a useful alternative for the identification of several NTM species, and a versatile tool for the characterization of clinical NTM isolates. At times it requires additional tests for definite species diagnosis and correct identification. Copyright © Apmis 2007.