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The analysis by Denaturing Gradient Gel Electrophoresis (DGGE) of the PCR-amplified V3 region of 16S rRNA gene was previously shown to detect and differentiate a large number of human and animal mycoplasmas. In this study, we further assessed the suitability of the technique for epidemiological surveillance of mycoplasmas belonging to the ‘Mycoplasma mycoides’ cluster, a phylogenetic group that includes major ruminant pathogens.The V3 region of 16S rRNA genes from approx. 50 field strains was amplified and analysed by DGGE. Detection and identification results were compared with the ones obtained by antigenic testing and sequence analysis.The DGGE technique is robust and valuable as a first-line test, but the patterns obtained for strains belonging to the ‘M. mycoides’ cluster were too variable within a taxon and in contrast too conserved between taxa to allow an unequivocal identification of isolates without further analysis.Issues raised by the quest for a single universal test able to detect and identify any mycoplasma in one clinical sample are thoroughly documented.