This retrospective study sought to systematically identify clinical and radiological features differentiating Mycobacterium kansasii from Mycobacterium tuberculosis infection. The sample included matched patients with a culture-positive diagnosis of M. tuberculosis infection (n = 121) or M. kansasii infection (n = 62) derived from the databases of two tuberculosis centers. Data on patient background and clinical features were collected, and chest radiographs were analyzed. Sixty percent of the M. kansasii group were native Israelis compared to 15% of the M. tuberculosis group (p = 0.0001). M. tuberculosis infection was associated with a higher rate of human immunodeficiency virus (HIV) infection (p = 0.03) and M. kansasii infection with a higher rate of lung disease (p = 0.0001). M. tuberculosis infection was characterized by a higher likelihood of bilateral disease (p = 0.005), pleural effusions, and lymphadenopathy (p = 0.006 and p = 0.001, respectively). There were ten deaths, all in the M. tuberculosis group. On multivariate logistic regression, the presence of chronic obstructive pulmonary disease and associated lung disease were significant predictors of M. kansasii infection. The findings show that there are group differences between the clinical features of the two infections. In the setting of pulmonary mycobacterial infection, the presence of coinfection with HIV, bilateral disease, pleural effusion, and lymphadenopathy make M. kansasii infection very unlikely.