Strain characterization of Mycobacterium tuberculosis has been based mainly on mycobacteriophage typing or chromosomal DNA restriction fragment analysis. In this study 10 randomly selected EcoRI chromosomal DNA fragments of M. tuberculosis H37RV were labelled with digoxigenin and used to probe the Southern blot preparation of EcoRI or BstEII digested chromosomal DNA of clinical isolates of M. tuberculosis. 2 probes were able to reveal restriction fragment length polymorphism. Each of the probes divided 15 pulmonary and 6 cerebrospinal fluid (CSF) isolates of M. tuberculosis into 3 groups and combination of both probes divided them into 4 groups. All of the 6 CSF isolates belonged to 1 group while only 5 of the 15 pulmonary isolates belonged to the same group. Work is continuing in order to characterize the nature of the probe and confirm the results in a larger population.