Abstract: Objective To analyze the clinical and bacteriological characteristics of pulmonary infection of Mycobacterium septium and discuss the differential diagnosis of pulmonary M. spetium infection. Methods The analysis was conducted on the clinical manifestations and laboratory detection results of a patient with pulmonary M. spetium infection. The identification of isolated strain was conducted with L-J, TCH/PNB differential media and multi-locus PCR, and the sequencing of rpoB/hsp65 gene of the strain was performed with PCR. Results The sputum smear and culture results were all positive for the patient. The identification result indicated that the strain was non-tuberculosis Mycobacterium (NTM). The rpoB/hsp65 gene of the strain shared 99% homology with M. spetium (ATCC 700731). The drug susceptibility test showed that the strain was resistant to most antibiotics, but sensitive to amikacin, ciprofloxacin, ofloxacin, levofloxacin, kanamycin and sulfamethoxazole. Conclusion This was the first case of pulmonary M. spetium infection diagnosed in China.