Introduction. Mycobacteria have a complex cell wall structure that includes many lipids which are often species specific. Besides giving a phenotypic signature these lipids are often involved in infectious processes of pathogenic mycobacteria by interfering with the host immune system. The biosynthesis pathway of the simplest glycopeptidolipids (GPLs) is relatively well understood and involves more than fifteen genes. Even though M. avium subsp. paratuberculosis (Map), the causative agent of Johne’s disease in cattle and other ruminants, produces a lipopeptide rather than GPL, its genome contains nevertheless a locus highly similar to the GPL biosynthetic pathway of M. avium subsp.avium (Mav). We showed that the module composition of the non-ribosomal protein synthase (Nrp) of Map, the enzyme involved in the synthesis of the peptidyl moiety, is dramatically different from that of other GPL producers such as M. smegmatis (Ms) and Mav. While Map isolates do not produce GPLs, they do produce lipopeptides without the carbohydrate moiety. However, the picture is not as clear regarding the diversity of lipopeptides produced among two lineages classified as type I/III or S-type (ovine) and type II or C-type (bovine) Map strains that have emerged from the common ancestor, M. avium subsp. hominissuis. The S-type isolates are readily distinguishable from C-type isolates based on genome studies and readily discriminated by genotyping methods. In addition to the genotypic distinctions between S- and C-type strains, phenotypic differences have been documented. The objective of this study was to identify the composition of lipopeptides in the S- type strains of Map and determine if they are different from the C-type strains Material and Method. To provide a genomic basis for the synthesis of the diversity of lipopeptides in Map strain S397, its recently published genome sequence was explored using in silico methods and completed by biochemical investigations. Results. Genetic characterization allowed us to predict the production of different lipopeptide components, depending on the strain type. This strategy showed that a nonribosomal peptide synthase, encoded by mps1, contains three amino acid specifying modules in all ovine strains analyzed, compared to five modules in bovine strains (C-type). Sequence analysis predicted these modules would produce the tripeptide Phe-N-Methyl-Val-Ala with a lipid moiety, termed lipotripeptide (L3P). Comprehensive physico-chemical analysis of Map S397 extracts confirmed the structural formula of the native L3P as D-Phe-N-Methyl-L-Val-L-Ala-OMe attached in N-ter to a 20-carbon fatty acid chain. Conclusion These findings add new phenotypic evidence that contribute to separate the Map S type to the C type. Furthermore deciphering the biosynthesis pathway of cell wall lipopeptides should contribute to better understand the determinants of the adaptation of a pathogen to a specific host but also the factors favoring transmission to a new host.