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Background: Multidrug-resistant (MDR) tuberculosis, defined as a disease caused by Mycobacterium tuberculosis strains, which are resistant to more antituberculous drugs (at least to isoniazid and rifampicin), is a problem frequently discussed in the Czech Republic. Cases of specific disease refractive to causal antituberculous therapy are associated with the risk of the spread of the causative agent among the population., Methods and Results: The National Reference Laboratory for Mycobacteria collected 2813 Mycobacterium tuberculosis strains isolated by Czech mycobacteriological laboratories in 1999 to 2001. All strains were tested for susceptibility to basic antituberculous drugs and then the MDR strains were further tested for susceptibility/resistance to other antituberculous and antibacterial drugs. The MDR strains were studied by DNA analysis (DNA fingerprinting restriction analysis, RFLP-Restriction Fragment Length Polymorphism) as well. Thirty-nine patients who had MDR tuberculosis were excretors of 56 Mycobacterium tuberculosis strains. In average, MDR tuberculosis accounted for 1.96% (1.7-2.4) of all cases of bacillary tuberculosis. The most frequent type of the multidrug resistance was that resistant to four basic antituberculous drugs (isoniazid, rifampicin, ethambutol and streptomycin). It was confirmed in 48.2% multidrug resistant strains., Conclusions: Isepamicin, clofazimin, capreomycin and amikacin are considered to be the most promising antituberculosis drugs. Based on RFLP profiles, 61.5% of strains were placed into 8 clusters while the other strains remained unclustered. No significant differences in geographical distribution and population structure were found between the excretors of clustered strains and those of unclustered strains. Preliminary comparison with restriction profiles of the MDR Mycobacterium tuberculosis strains in the international database suggests the uniqueness of Czech strains showing the profiles not found elsewhere to date.