The identification of an emerging pathogen in humans can remain difficult by conventional methods such as enrichment culture assays that remain highly selective, require appropriate medium and cannot avoid misidentifications, or serological tests that use surrogate antigens and are often hampered by the level of detectable antibodies. Although not originally designed for this purpose, the implementation of polymerase-chain-reaction (PCR) has resulted in an increasing number of diagnostic tests for many diseases. However, the design of specific molecular assays relies on the availability and reliability of published genetic sequences for the target pathogens as well as enough knowledge on the genetic diversity of species and/or variants giving rise to the same disease symptoms. Usually designed for clinical isolates, molecular tests are often not suitable for environmental samples in which the target DNA is mixed with a mixture of environmental DNA. A key challenge of such molecular assays is thus to ensure high specificity of the target genetic markers when focusing on clinical and environmental samples in order to follow the dynamics of disease transmission and emergence in humans. Here we focus on the Buruli ulcer (BU), a human necrotizing skin disease mainly affecting tropical and subtropical areas, commonly admitted to be caused by Mycobacterium ulcerans worldwide although other mycolactone-producing mycobacteria and even mycobacterium species were found associated with BU or BU-like cases. By revisiting the literature, we show that many studies have used non-specific molecular markers (IS2404, IS2606, KR-B) to identify M. ulcerans from clinical and environmental samples and propose that all mycolactone-producing mycobacteria should be definitively considered as variants from the same group rather than different species. Importantly, we provide evidence that the diversity of mycolactone-producing mycobacteria variants as well as mycobacterium species potentially involved in BU or BU-like skin ulcerations might have been underestimated. We also suggest that the specific variants/species involved in each BU or BU-like case should be carefully identified during the diagnosis phase, either via the key to genetic identification proposed here or by broader metabarcoding approaches, in order to guide the medical community in the choice for the most appropriate antibiotic therapy.