<p>Nucleotide primers used in this study.</p>

Bats play important roles in ecosystem functioning and host a remarkable diversity of blood parasites, including a limited number of bat-associated Trypanosoma species that have been described from Africa. Cave roosts provide critical habitat for bats and often support large, multispecies aggregations that may influence parasite transmission dynamics. Nevertheless, the diversity and host associations of trypanosomes within these environments remain poorly characterized, particularly in West Africa. To address this gap, we screened blood and ectoparasite samples from 57 bats representing ten species captured in nine caves located 0.4–2 km from human residences, as well as from one residential building in southeastern Nigeria, using PCR. Trypanosome DNA was detected in 22/57 bats (38.6%) and in six bat species across four families: Rousettus aegyptiacus (1/2), Hipposideros abae (11/23), H. jonesi (1/1), H. ruber (5/11), Rhinolophus landeri (2/10), and Mops condylurus (2/6). Phylogenetic analyses placed the trypanosome sequences within the Trypanosoma livingstonei species group, a clade of African bat-associated trypanosomes representing early-diverging lineages within the Trypanosoma cruzi clade, which includes the causative agent of Chagas disease. The Nigerian trypanosome sequences were not recovered as a single homogeneous lineage, and species delimitation analysis recovered them together with other T. livingstonei/ T. cf. livingstonei references across several putative molecular lineages. The exclusive detection of parasites of the T. livingstonei species group across multiple bat species indicates low trypanosome diversity within this cave system but reveals phylogenetic structuring within this parasite group. The findings highlight the need for broader geographic sampling, multilocus data, and ecological measurements to clarify the drivers of trypanosome diversity in cave-dwelling bats.