The techniques of spoligotyping and mycobacterial interspersed repetitive unit and variable-number tandem repeat typing with 24 loci (MIRU-VNTR-24), have been used to study the molecular epidemiology of tuberculosis. The aim of this study was: to evaluate the discriminative power of MIRU-VNTR 24 loci alone and in association with spoligotyping in clinical isolates of M tuberculosis in Venezuela; the allelic diversity of the 24 loci; and the discriminative power for the combination of 24 and 15 loci, 12 traditional loci (12t), those with higher allelic diversity and a new combination named 12inv. We analyzed one set of 104 strains of different lineages and a second set of 431 strains belonging to the Latin-America and Mediterranean lineage (LAM) that is predominant in Venezuela. The determination of allelic diversity showed that 4052, 2163b, 424 y 2996 are highly discriminative. Clustering rates of MIRU-VNTR 24 loci, spoligotyping and MIRU-VNTR combined with spoligotyping for 104 isolates were 18.27%, 71.15% and 14.4%, respectively, whereas with the 431 LAM strains the values were 43.2 %, 95.8% and 37.4%. MIRU-VNTR combinations of 15, 12inv and 4 loci were more discriminatory than 12t. Clustering rates for MIRU-VNTR 15 and 12inv loci coupled with spoligotyping in the 104 isolated was 21% and 23%, while for LAM strains was 52% and 46% respectively. The number of different genetics patterns for 15 and 12inv loci were similar. In conclusion, we propose the use of a small number of informative loci MIRU-VNTR coupled to spoligotyping to investigate the transmission of tuberculosis in Venezuela.