BACKGROUND: Female frog-biting midges (Corethrella) are hematophagous micropredators that feed on frogs and serve as vectors for trypanosomes (Trypanosoma), unicellular flagellate parasites. Little is known about the infection ecology and host specialization within this tritrophic interaction. METHODS: In this study, we explore the prevalence, diversity and specificity of Trypanosoma infections in Corethrella across various localities in tropical America and Borneo by sequencing both midge and trypanosome markers in midge samples. RESULTS: Bayesian phylogenetic analyses and ASAP species delimitation of Corethrella (cytochrome c oxidase I [COI]) and Trypanosoma (18S, glyceraldehyde 3-phosphate dehydrogenase [GAPDH]) revealed a previously unknown high diversity of frog-biting midge-associated trypanosomes. Across regions and localities, the infection prevalence in midges caught by acoustic midge traps ranged from 2.9% to 23.5%, suggesting that a notable proportion of midges carried trypanosomes, likely acquired from a previous blood meal. At one locality, La Gamba (Costa Rica), the infection prevalence in trap-caught midges was 10.9%, while it was even higher in midges collected directly from frog hosts (20.7%), in agreement with the hypothesis that midges ingest trypanosomes from infected frogs. Bipartite network analyses revealed high degrees of specialization of Trypanosoma in trap-caught Corethrella, both across all localities (H2' = 0.87) and when analyzed for our most sampled locality (Cahuita, Costa Rica) alone (H2' = 0.94). CONCLUSIONS: Our data suggest that most trypanosomes detected in trap-caught midges are established, host-competent (i.e., specialist) parasites in an infective stadium.