Buruli ulcer is a neglected skin disease caused by Mycobacterium ulcerans (MU), and affects 1000 people each year worldwide and particularly in African countries. Eradication of Buruli ulcer is difficult because of the lack of early diagnostic in rural endemic regions, and the unknown of the disease in national health care system. In the rural wetlands and marshes in Central and West Africa, children are most affected. MU belongs to environmental Mycolactone Producing Mycobacteria (MPMs) and presents high genetic diversity of the circulating strains. Diagnoses were applied by culture and genome detection by Polymerase Chain Reaction (PCR). The PCR became a gold method to confirm clinical and environmental samples for MU. The MIRU-VNTR method and the Restriction Fragment Length Polymorphism (RFLP) combined with MIRU-VNTR markers were used to discriminate Mycobacteria from different sources. The aim of this study was to investigate the molecular diversity of different mycobacteria sources, from environment, culture strains, and clinical samples by using combined MIRU-VNTRTyping and RFLP. A total of 26 samples (water, sediment, mycobacteria strains, and swab) from endemic sites were first confirmed by IS2404 or Ziehl-Neelsen staining. The samples were analyzed by PCR typing for 4 specific markers (MIRU-1, VNTR6, VNTR19, ST-1) and the amplicons were digested with MspI restriction endonuclease and separated by 3% agarose gel electrophoresis for PCR-RFLP analysis. Our results showed different amplification by VNTR-MIRU-typing. For environmental samples a low amplification was detected by 25% for PCR-MIRU-1. Culture strains and clinical samples had amplification rate of 35.7% and 62.5% respectively. ST-1 had the best amplified marker for culture strains by 71.4%, while clinical samples have good amplification rate by 62.5 % for all markers. PCR-RFLP-profiles of clinical samples were identical, while environmental and mycobacteria strains showed different PCR-RFLP-profiles. We have developed a PCR-RFLP sensitive, easier and inexpensive approach to confirm genotyping of nontuberculosis mycobacteria in endemic countries for environmental screening. We suggest mutation in repeat loci of VNTR-MIRU sequence and the adaptation of mycobacteria from environment to human. This study confirms the circulation of several genotypes of mycobacteria in Ivory Coast.