Early identification of mycobacterial species is crucial for early diagnosis. PCR-multiplex method performed on randomly chosen 54 mycobacteria isolates originating from clinical samples was found to be an inexpensive, quick and reliable alternative for commercially available diagnostics tests. Although the results of gene probes identification performed by NTLDR were generally consistent with multiplex PCR, two mixed Mycobacterium bovis BCG/Mycobacterium tuberculosis infections and a single misdiagnosis of M. tuberculosis with M. bovis were found. The routine application of multiplex-PCR has the potential to make diagnostics surveillance studies feasible.