peer reviewed ; The tuberculosis (TB) incidence rate in Brussels-Capital Region is 3-fold higher than in Belgium as a whole. Eight years after the realization of initial prospective population-based molecular epidemiology investigations in this Region, a similar study over the period 2010-2013 was conducted. TB strains isolated from 945 patients were submitted to genotyping by standardized 24-locus-MIRU-VNTR typing and spoligotyping. The phylogenetic analysis showed that the LAM (16.7%) and Haarlem (15.7%) branches are the two most prevalent TB lineages circulating in Brussels. Analysis of the MDR subgroup showed an association with Beijing strains (39.9%) and patients native of Eastern Europe (40.7%). Genotyping detected 113 clusters involving 321 patients, giving a recent transmission index of 22.9%. Molecular-guided epidemiological investigations and routine surveillance activities revealed family transmission or social contact for patients distributed over 34 clusters. Most of the patients were foreign-born (75.7%). However, cluster analysis revealed only limited transnational transmission. Comparison with the previous study shows a stable epidemiological situation except for the mean age difference between Belgian-born and foreign-born patients which has disappeared. This study confirms that molecular epidemiology has become an important determinant for TB control programs. However, sufficient financial means need to be available to perform all required epidemiological investigations. © 2017 Vluggen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.