Trypanosomes are blood-borne parasites infecting a range of mammalian hosts worldwide. In Australia, an increasing number of novel Trypanosoma species have been identified from various wildlife hosts, some of which are critically endangered. Trypanosoma noyesi is a recently described species of biosecurity concern, due to a close relationship to the South American human pathogen, Trypanosoma cruzi. This genetic similarity increases the risk for introduction of T. cruzi via a local vector. Unfortunately, there is a lack of knowledge concerning the vectorial capacity of Australian invertebrates for native Trypanosoma species. Australian ixodid ticks (Ixodidae), which are widespread ectoparasites of mammalian wildlife, have received the most attention as likely candidates for trypanosome transmission and have been previously implicated as vectors. However, as all studies to date have focused on blood-fed ticks collected directly from infected mammalian hosts, the question of whether ticks maintain a trypanosome infection between blood meals is unknown. In this study, we investigated the presence of Trypanosoma within 148 Australian adult and nymph questing ticks of the species Amblyomma triguttatum, Ixodes australiensis, Ixodes myrmecobii and larvae Ixodes spp., collected from an endemic region of south-west Australia. Using a novel HRM-qPCR detection method that can discriminate between species of Trypanosoma based on primer melting temperature (T-m), we report the first molecular detection of Trypanosoma DNA in Australian questing ticks, with 6 ticks DNA positive for T. noyesi. Additionally, the presence of intact T. noyesi parasites within all (n = 3) smeared gut and gland contents of questing ticks was confirmed using a fluorescence in situ hybridisation (FISH) assay. Whilst this study was unable to determine the in situ tissue location of trypanosomes for the purpose of discerning a potential route of transmission, these combined molecular and FISH smear data indicate that trypanosomes can persist in ticks between blood meals and that ticks are possibly vectors in the transmission of T. noyesi between native wildlife. Transmission experiments are still required to evaluate the competency of Australian ticks as vectors for T. noyesi. Nevertheless, these novel findings warrant further investigation concerning potential life stages and the development of trypanosomes in both Australian, and other, tick species.