Spoligotyping is a major tool for molecular typing of Mycobacterium tuberculosis complex organisms. For epidemiological purposes, strains are considered clonal only when their spoligotyping patterns are identical. We report a change in the spoligotyping profiles of truly isogenic strains (a clinical isolate and a subculture derived in the laboratory) caused by deletion of a direct variable repeat. Without the information about the relationship between them, a link between these strains would have gone unnoticed. Evolutionary events should be taken into account in the interpretation of spoligotyping results and in the design of databases.