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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. This study investigated the distribution of Mtb mutations and spoligotypes in rural Eastern Cape Province. The Mtb isolates included were 1157 from DR-TB patients and analysed by LPA followed by spoligotyping of 441 isolates. The distribution of mutations and spoligotypes was done by spatial analysis. The rpoB gene had the highest number of mutations. The distribution of rpoB and katG mutations was more prevalent in four healthcare 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 a better picture of distribution. [ABSTRACT FROM AUTHOR]