Several methods are available for the molecular typing of Mycobacterium tuberculosis complex isolates. The results of the recent research demonstrated that Mycobacterial Interspersed Repetitive Unit (MIRU)-Variable Number Tandem Repeats (VNTR) method has high discriminatory power and reproducibility, is easy to perform, and available for multi-center studies and automation. However, there is insufficient data about the MIRU-VNTR profiles in Turkey. The aim of this study was to determine the most appropriate MIRU-VNTR combinations to distinguish cross contaminations and nosocomial infections in routine mycobacteriology laboratory practice. Following molecular typing of 152 clinical isolates which were consecutively isolated from different patients in two years period (August 2004-July 2006) in our laboratory, a retrospective analysis of MIRU-VNTR data of 12 loci primers was performed by an "in-house" computer based programme. The programme was prepared by using Microsoft QuickBASIC programming language and all of the data were calculated by the help of this programme. The best combinations to differentiate the clusters and to identify the unique isolates were determined out of 4095 possible results of 12 different primer pairs. According to our 152 MIRU-VNTR results, to determine cross contaminations and nosocomial infections in routine mycobacteriology laboratory practice, we recommend to use primers 26, 40, 16, 10 and 23 in the first step; primers 31, 27, 20 and 2 in the second step, and primers 4, 24 and 39 in the third step. The created software is user friendly, fast and meets the requirements of routine clinical mycobacteriology laboratories. Besides its discriminatory power, the speed and cost-effectiveness of a typing method is also considerable. According to the results of this study it was suggested that for more rapid and economic molecular typing of M.tuberculosis and related epidemiological investigations, MIRU-VNTR should be performed in a stepwise manner.