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Invasive <i>Rattus</i> species are carriers of haemotropic <i>Mycoplasmas</i> (haemoplasmas) globally, but data from Africa are lacking. Using a PCR-sequencing approach, we assessed haemoplasma prevalence and diversity in kidney and buccal swabs collected from three invasive <i>Rattus</i> species (<i>Rattus rattus</i>, <i>R. norvegicus</i> and <i>R. tanezumi</i>) in Gauteng Province, South Africa. Whilst the overall sequence-confirmed haemoplasma prevalence was 38.4%, infection rates in <i>R. rattus</i> (58.3%) were significantly higher (χ² = 12.96; df = 2; n = 99 <i>p</i> < 0.05) than for <i>R. tanezumi</i> (14.3%). Differences between host sex (χ² = 3.59 × 10⁻³¹; df = 1; n = 99; <i>p</i> = 1.00) and age (χ² = 4.28; df = 2; n = 99; <i>p</i> = 0.12) were not significant. Whilst buccal (1.01%) and ectoparasite positivity (2.13%) were low, these results suggest that multiple transmission routes are possible. Three phylogenetically distinct lineages, consistent with global rat-associated strains described to date, were detected, namely, ‘<i>Candidatus</i> Mycoplasma haemomuris subsp. Ratti’, and two Rattus-specific haemoplasmas that are yet to be formally described. These results expand the known distribution of invasive rat-associated haemoplasmas and highlight the potential for pathogen co-invasion of new territories together with invading rodent hosts.