Mycobacterium avium and Mycobacterium paratuberculosis are primary pathogens for animals. They belong to the same complex, but are no longer considered as separate species. Differences are based on their growth characteristics, mycobactin requirement, and the natural host-range; although they have different IS sequences, the overall similarity at the genome level justifies the lumping in one species. Among the innovative identification methods, besides genome-based techniques, there is High Performance Liquid Chromatography (HPLC) analysis of cell wall mycolic acids. We have previously shown the usefulness of this new technique for the species identification of mycobacteria, and we have shown the typical profile of M. avium. M. paratuberculosis strains (the reference ATCC 19698 and animal isolates) were extracted, and the mycolic acid profiles obtained by HPLC were compared to the profile from M. avium. The HPLC mycolic acid profile of M. paratuberculosis strains were very similar to the M. avium profile. Therefore, the technique is not suitable for a rapid species identification of M. paratuberculosis. Nevertheless, our data support the view that M. avium and M. paratuberculosis be considered utmost as subspecies, and confirm HPLC analysis of mycolic acid profiles as the technique most similar to 16S rRNA sequencing or DNA-DNA hybridization.