Objective To investigate the clinical and CT features of spondylitis caused by non-tuberculous mycobacteria (NTM) and Mycobacterium tuberculosis (TB). Methods The clinical information and CT of 63 gender- and age-matched patients with microbiologically confirmed NTM (21) and TB (42) spondylitis in our hospital from 2009 to 2022 were retrospectively analyzed. Results The majority of NTM spondylitis was middle-aged to elderly women. The incidence of trauma (38.10%), disease severity (47.62%), extraspinal bone involvement (76.19%), skin and soft tissue abscess (61.90%), and CD4 +T cell reduction (38.10%) in NTM group was significantly higher (P=0.001, 0.036, <0.001, <0.001, 0.014) than those in TB group (4.76%, 19.05%, 19.05%, 4.76%, 9.52%). The number of infected vertebrae (5, 2.5-9), incidence of central (90.48%) or peripheral (23.81%) distribution of lesions within the vertebrae, more than 1 lesion in the same vertebrae (80.95%), predominant sclerotic mixed bone destruction (76.19%), nodular or patchy increased density (61.90%), and thick ring sign (38.10%) in NTM group were significantly higher (P=0.003, <0.001, 0.013, <0.001, 0.003, <0.001, <0.001) than that of TB group was (2, 2-4; 11.90%; 2.38%; 1.52%; 35.71%; 0; 0). The incidence of lesions involving only two adjacent vertebrae (52.38%), subligamentous lesions (95.24%), dead bone (64.29%), vertebral collapse (40.48%), spinal deformity (73.81%), intervertebral space stenosis (4.76%), calcification in paravertebral abscess (100%), peripheral muscle abscess (45.24%), the thickness of paravertebral soft tissue swelling/abscess (1.45 cm, 1-1.9 cm), and the number of vertebrae surrounded by paravertebral abscess (3, 2-4) in TB group were significantly higher (P=0.006, 0.006, 0.003, <0.001, 0.015, 0.003, <0.001, <0.001, 0.024, <0.001, <0.001) than that of NTM group (14.29%; 9.52%; 28.57%; 19.05%; 28.57%; 14.29%; 0.5 cm, 0.2-0.95 cm; 1, 1-2). Conclusion The clinical and CT features are helpful for differential diagnosis of NTM and TB spondylitis. [ABSTRACT FROM AUTHOR]