Toll-like receptors (TLRs) are evolutionary conserved in all animal species and they play key roles in recognising and binding a wide range of pathogens. At least twelve TLRs have been observed in mammals and they can be divided into viral and non-viral TLRs according to their ligand recognition. The aim of this study was to characterize the levels of genetic diversity at TLRs in two bottlenecked Australian native marsupial species, the Tasmanian devil (Sarcophilus harrisii), and the koala (Phascolarctos cinereus). In this study, we have identified ten TLRs (TLR2-10 and TLR13) in the devil and nine (TLR2-10) in the koala. TLR13 ortholog couldn’t be found in the koala sequence transcriptome. The TLRs protein sequence similarity with human is on an average of 60% in the devil and 64.17% in the koala. The devils TLR4, TLR5, TLR7, TLR8, TLR9, TLR10 and TLR13 are monomorphic and TLR2, TLR3 and TLR6 are harbouring only two alleles in the 25 study samples. With the nine koala TLRs, TLR10 is monomorphic and 2 to 6 alleles have been observed varied at other TLRs in the 20 koalas from the NSW population. Future studies will focus on TLRs diversity and disease status in these species.