The aim of this study was to assess the feasibility of a method for presumptive identification of mycobacteria, based on the morphology in smears prepared from radiometric Bactec-positive cultures (Becton Dickinson, USA) and to select the appropriate DNA probe (AccuProbe; Gen Probe, USA). The smear morphology of acid-fast bacilli was evaluated in 468 positive cultures from clinical samples: 313 Mycobacterium tuberculosis complex, 67 Mycobacterium avium complex, 32 Mycobacterium kansasii, 49 Mycobacterium xenopi, and seven Mycobacterium gordonae. The sensitivity and specificity for various morphological patterns were as follows: cord formation for Mycobacterium tuberculosis complex 90% and 100%, respectively; striped bacilli for Mycobacterium kansasii, 66% and 99%; sea urchin for Mycobacterium xenopi, 96% and 99%; short bacilli for Mycobacterium avium complex, 61% and 99%; fine-striped bacilli associated with Mycobacterium avium complex from blood samples, 33% and 98%. This criterion was applied in the selection of a suitable DNA probe for the identification of 178 cultures. The correct probe was selected in 98%, 97%, and 72% of cultures, respectively, for Mycobacterium tuberculosis complex, Mycobacterium avium complex, and Mycobacterium kansasii. The observation of acid-fast bacilli morphology in radiometric cultures is a rapid and cost-efficient method for presumptive identification of common clinical isolates of mycobacteria.