Background <p>Female frog-biting midges (<i>Corethrella</i>) are hematophagous micropredators that feed on frogs and serve as vectors for trypanosomes (<i>Trypanosoma</i>), unicellular flagellate parasites. Little is known about the infection ecology and host specialization within this tritrophic interaction.</p> Methods <p>In this study, we explore the prevalence, diversity and specificity of <i>Trypanosoma</i> infections in <i>Corethrella</i> across various localities in tropical America and Borneo by sequencing both midge and trypanosome markers in midge samples.</p> Results <p>Bayesian phylogenetic analyses and ASAP species delimitation of <i>Corethrella</i> (cytochrome c oxidase I [COI]) and <i>Trypanosoma</i> (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 <i>Trypanosoma</i> in trap-caught <i>Corethrella,</i> both across all localities (H2′ = 0.87) and when analyzed for our most sampled locality (Cahuita, Costa Rica) alone (H2′ = 0.94).</p> Conclusions <p>Our data suggest that most trypanosomes detected in trap-caught midges are established, host-competent (i.e., specialist) parasites in an infective stadium.</p> Graphical Abstract <p></p>