Abstract Whole genome sequencing (WGS) is able to differentiate closely related Mycobacterium tuberculosis variants within the same transmission cluster. Our aim was to evaluate if this higher discriminatory power may help identify and characterize more actively transmitted variants and understand the factors behind their success. We selected a robust MIRU-VNTR-defined cluster from Almería, Spain (22 cases throughout 2003–2019). WGS allowed discriminating, within the same epidemiological setting, between a successfully transmitted variant and seven closely related variants that did not lead to secondary cases, or were involved in self-limiting transmission (one single secondary case). Intramacrophagic growth of representative variants was evaluated in an in vitro infection model using U937 cells. Intramacrophage multiplication ratios (CFUs at Day 4/CFUs at Day 0) were higher for the actively transmitted variant (range 5.3–10.7) than for the unsuccessfully transmitted closely related variants (1.5–3.95). Two SNPs, mapping at the DNA binding domain of DnaA and at kdpD , were found to be specific of the successful variant.