BackgroundHaemosporidian parasites of the genusPolychromophilusinfect bats worldwide. They are vectored by obligate ectoparasitic bat flies of the family Nycteribiidae. Despite their global distribution, only fivePolychromophilusmorphospecies have been described to date. The two predominant species,Polychromophilus melanipherusandPolychromophilus murinus, are broadly distributed and mainly infect miniopterid and vespertilionid bats, respectively. In areas where species from different bat families aggregate together, the infection dynamics and ability of eitherPolychromophilusspecies to infect other host families is poorly characterized.MethodsWe collected 215 bat flies from two bat species,Miniopterus schreibersiiandRhinolophus ferrumequinum, which sometimes form mixed clusters in Serbia.Miniopterus schreibersiiis known to be frequently infected withP. melanipherus, whereasR. ferrumequinumhas been observed to be incidentally infected with bothPolychromophilusspecies. All flies were screened forPolychromophilusinfections using a PCR targeting the haemosporidiancytbgene. Positive samples were subsequently sequenced for 579 bp of cytochrome b (cytb) and 945 bp of cytochrome oxidase subunit 1 (cox1).ResultsPolychromophilus melanipherusDNA was detected at six out of nine sampling locations and in all three examined bat fly species collected fromM. schreibersii(Nycteribia schmidlii,n= 21;Penicillidia conspicua,n= 8;Penicillidia dufourii,n= 3). Four and five haplotypes were found forcytbandcox1, respectively. Evidence for multiplePolychromophilushaplotypes was found in 15 individual flies. These results point to a high diversity ofP. melanipherusparasites inMiniopterushosts and efficient transmission throughout the study area. A singlePhthiridium biarticulatumbat fly collected fromR. ferrumequinumscreened positive forP. melanipherus, but only yielded a partialcox1sequence fragment. Nevertheless, this result suggests that secondary hosts (both bat and fly species) are regularly confronted with this parasite.ConclusionsThe results of this study provide new insights into the prevalence and distribution ofPolychromophilusparasites in European bats and their nycteribiid vectors. The use of bat flies for the non-invasive investigation ofPolychromophilusinfections in bat populations has proven to be efficient and thus represents an alternative for large-scale studies of infections in bat populations without the need to invasively collect blood from bats.Graphical AbstractSupplementary InformationThe online version contains supplementary material available at 10.1186/s13071-023-05786-1.