Central Asia Outbreak (CAO) clade is a branch of the Mycobacterium tuberculosis Beijing genotype that is associated with multidrug-resistance, increased transmissibility and epidemic spread in parts of the former Soviet Union. Furthermore, migration flows bring these strains far beyond their areas of origin. We aimed to find a specific molecular marker of the Beijing CAO clade and develop a simple and affordable method of its detection. Based on the bioinformatics analysis of the large M. tuberculosis whole-genome sequencing (WGS) dataset (n=1398), we identified IS6110 insertion in the Rv1359-Rv1360 intergenic region as a specific molecular marker of the CAO clade. We further designed and optimized a multiplex PCR method to detect this insertion. The method was validated in silico with recently published WGS dataset from Central Asia (n=277) and experimentally, with M. tuberculosis isolates from European and Asian parts of Russia, former Soviet Union and East Asia (n=319). The developed molecular assay may be recommended for rapid screening of retrospective collections and for prospective surveillance, when comprehensive but expensive WGS is not available or practical. The assay may be especially useful in the high MDR-TB burden countries of the former Soviet Union and in the countries with respective immigrant communities.