To identify mycobacteria isolated from sputa of a 51-year-old female and a 72-year-old male patient with pneumoconiosis.Mycobacteria species were isolated from sputa of a 51-year-old female. The culture was always negative in spite of positive smears before the final isolation in 1988. A 72-year-old male patient suffered from pneumoconiosis and the acid-fast bacillus was isolated by routine sputum examination in 2003. These two strains of acid-fast bacilli were identified as Mycobacterium heckeshornense by partial sequencing of 16S rRNA and rpoB and conventional methods (biochemical and routine culture methods).These two strains grew on 1% Ogawa's slant medium at 37 degrees C and 42 degrees C, but not at 28 degrees C. They formed yellowish colonies in the dark (Scotochromogen). They were classified as a slowly growing Mycobacteria. As it was difficult to distinguish M. heckeshornense from M. xenopi by conventional methods including growth rate, temperature range of mycobacterial growth, light coloration reaction, biochemical and biological tests, virulence using guinea pigs and drug susceptibility test were further explored. Finally two were identified as M. heckeshornense by summing of these results.Mycobacteria species that grow at 42 degrees C for four weeks, imply M. xenopi with a DDH method. It is essential to perform both sequencing of 16S rRNA and rpoB gene and a biochemical method for the purpose of distinguishing M. heckeshornense from M. xenopi.