ABSTRACTMycobacterium ulcerans, an environmental opportunistic pathogen, causes necrotic cutaneous and subcutaneous lesions, named Buruli ulcers, in tropical countries. PCR-derived tests used to detectM. ulceransin environmental and clinical samples do not allow one-shot detection, identification, and typing ofM. ulceransamong closely relatedMycobacterium marinumcomplex mycobacteria. We established a 385-memberM. marinum/M. ulceranscomplex whole-genome sequence database by assembling and annotating 341M. marinum/M. ulceranscomplex genomes and added 44M. marinum/M. ulceranscomplex whole-genome sequences already deposited in the NCBI database. Pangenome, core genome, and single-nucleotide polymorphism (SNP) distance-based comparisons sorted the 385 strains into 10M. ulceranstaxa and 13M. marinumtaxa, correlating with the geographic origin of strains. Aligning conserved genes identified onePPE(proline-proline-glutamate) gene sequence to be species and intraspecies specific, thereby genotyping the 23M. marinum/M. ulceranscomplex taxa. PCR sequencing of thePPEgene correctly genotyped nineM. marinum/M. ulceranscomplex isolates among oneM. marinumtaxon and threeM. ulceranstaxa in the African taxon (T2.4). Further, successfulPPEgene PCR sequencing in 15/21 (71.4%) swabs collected from suspected Buruli ulcer lesions in Côte d’Ivoire exhibited positiveM. ulceransIS2404 real-time PCR and identified theM. ulceransT2.4.1 genotype in eight swabs andM. ulceransT2.4.1/T2.4.2 mixed genotypes in seven swabs.PPEgene sequencing could be used as a proxy for whole-genome sequencing for the one-shot detection, identification, and typing of clinicalM. ulceransstrains, offering an unprecedented tool for identifyingM. ulceransmixed infections.IMPORTANCEWe describe a new targeted sequencing approach that characterizes thePPEgene to disclose the simultaneous presence of different variants of a single pathogenic microorganism. This approach has direct implications on the understanding of pathogen diversity and natural history and potential therapeutic implications when dealing with obligate and opportunistic pathogens, such asMycobacterium ulceranspresented here as a prototype.