Tuberculosis (TB), an infectious airborne disease caused by Mycobacterium tuberculosis (Mtb), is a serious public health threat reported as the leading cause of morbidity and mortality worldwide. South Africa is a high TB burden country with TB being the highest infectious disease killer. The study investigated the distribution and clustering of Mtb mutations and spoligotypes in rural Eastern Cape Province. The Mtb isolates included were 1,157 from DR-TB patients and analysed by LPA followed by spoligotyping of 441 isolates, of these, 36 were whole genome sequenced. Distribution of mutations and spoligotypes was done by spatial analysis and clustering analysis was done by Bayesian model-based clustering of allele frequencies at heterozygous sites, using Mclust package in R. The rpoB gene had highest number of mutations. The distribution of rpoB and katG mutations was more prevalent in four health care facilities, inhA mutations were more prevalent in three healthcare facilities and heteroresistant isolates were more prevalent in five healthcare facilities. The Mtb was genetically diverse with Beijing more prevalent and largely distributed. Spatial analysis and mapping of gene mutations and spoligotypes revealed better picture of distribution. Clustering of isolates indicates that there is transmission of mixed infection in this area.