BACKGROUND: Most mammalian Plasmodium species possess distinct 18S ribosomal RNA (rRNA) gene copies, which are differentially expressed in vertebrate hosts and mosquito vectors. Although similar sequence patterns were found in avian haemosporidian parasites, expression patterns have not been studied yet. This study aimed to test whether 18S variants of Plasmodium relictum SGS1 and Haemoproteus tartakovskyi SISKIN1 are expressed differentially in bird hosts and dipteran vectors using real-time quantitative polymerase chain reaction (qPCR) and chromogenic in situ hybridization (CISH). METHODS: Eurasian siskins (Spinus spinus) experimentally infected with P. relictum SGS1 and naturally infected with H. tartakovskyi SISKIN1 were used. Culex quinquefasciatus mosquitoes (SGS1) and Culicoides nubeculosus biting midges (SISKIN1) were fed on the blood of infected birds and maintained for several days to allow for the development of oocysts and sporozoites. Total RNA was extracted from bird blood and a subset of the dipteran vectors during each stage of parasite development, followed by qPCRs specifically targeting distinct 18S variants of the two parasites. Organs of the donor birds and whole bodies of the vectors were examined histologically using CISH by targeting different 18S variants of the parasites. RESULTS: Plasmodium relictum SGS1 expressed two 18S variants in bird blood and mosquitoes, but their expression levels were reversed in birds and vectors, with one variant being preferentially expressed over the other. Using CISH, oocysts were stained with probes targeting both 18S variants, but sporozoites could not be detected, suggesting a suboptimal development of the parasites. Haemoproteus tartakovskyi SISKIN1, which features three distinct 18S variants, expressed one 18S variant in bird blood and two variants in the biting midges, while no signals were detected for the third variant. The results were corroborated by CISH, but surprisingly, some oocysts were also stained by the probe targeting the third variant. CONCLUSIONS: The results indicate that distinct 18S variants of the two parasite species are differentially expressed in bird hosts and vectors. Moreover, for the first time, we provide visualizations of avian haemosporidian oocysts in tissue sections of the vectors, with the discovery of extraintestinal development of oocysts in SISKIN1-infected biting midges.