Background: Fluorescence expression systems adapted to the analysis of host-mycoplasma interactions were recently developed. The aim of this work was to apply them to study the colonisation and persistence capabilities of the bovine pathogens Mycoplasma mycoides subsp. mycoides (Mmm) and Mycoplasma bovis in bovine cells. Methods. Mini-transposons affording high-level expression of GFP2, mCherry, mKO2 and mNeonGreen were used to mark Mmm and M. bovis strains. The resulting fluorescent mutants were characterised by epifluorescence microscopy and fluorimetry and the sites of transposon insertion were identified by sequencing. Interactions of mNeonGreen mutants with bovine cells were analysed using flow cytometry and confocal microscopy. Results: The production, selection and characterisation of fluorescent clones were straightforward and compatible with the production of fluorescent mutant banks. M. bovis presented much higher adhesion, invasion and proliferation capacities than Mmm in culture with non-phagocytic cells and showed increased resistance to elimination by macrophages. Conclusion: The remarkable differences in the invasion and persistence capabilities of Mmm and M. bovis observed here are in agreement with the pathogenesis of the infections caused by these mycoplasmas. mNeonGreen fluorescent mutants have proven extremely useful for analysis of mycoplasma-host cell interactions. Furthermore, the fluorescence expression tools used in this study offer innumerable perspectives for the functional analysis of a wide range of mycoplasma species both in vitro and in vivo.