Tuberculosis (TB) remains one of the most outstanding diseases in global health concern. It is caused by Mycobacterium tuberculosis Complex (MTBC) strains. ST 61, a cluster of the LAM10_CAM family has been shown to be the most prevalent cluster implicated in TB transmission in Cameroon. The present study is aimed at revealing the most discriminative loci applied to this cluster and investigating the genetic pattern in the ST 61 cluster clinical samples. This was a 12 month cross- sectional study that included 45 randomly selected clinical specimens previously collected during the CANTAM 1 project. Following DNA extraction, amplification of 12-locus Mycobacterial Interspersed Repetitive Unit-Variable Tandem Repeat (MIRU-VNTR) analysis (MIRU 40, ETR A, QUB 26, Mtub 39, QUB 4156, MIRU26, MIRU 31, MIRU 16, MIRU 02, MIRU 04, MIRU 23, and MIRU 27) PCR assay and gel electrophoresis were performed. The amplicon sizes, the allele assignations, the allelic pattern and the technic discriminative potential were determined using MIRU-VNTR plus web application and a dendogram was generated. 45% genetic pattern, and 66,6% clustering rate were reported. Overall, ETR A, MIRU 4, MIRU 16, MIRU 23, MIRU 27 revealed as the most discriminating loci applied to the cluster ST 61. The identification of the most discriminative locus ETRA, MIRU 4, MIRU 16, MIRU 23, and MIRU 27 applied to a precise cluster is a safe economic and time gain option to enhance the difference between strains at the cluster level.