Background The mobility of birds across or between continents exposes them to numerous vectors that have the potential to transmit pathogens and spread them into new regions. A combination of rich species diversity of birds along with the small amount of molecular studies in Iran makes observing the blood parasite distribution in wild avian populations indispensable for further estimation and administration of blood parasites. Methodology/Principal findings In order to evaluate the infection rate and molecular context of avian blood parasites, bird samples were collected (passerine = 316 and non-passerine = 14) in eight provinces of northern Iran between June to September 2015 and 2016. All bird samples were examined for haematoprotozoan infections by morphological screening using light microscope and mtDNA cytb gene amplification. A total of 115 birds were positive for blood parasites by molecular approach (34.84% overall infection). The infection rate of Haemoproteus, Plasmodium, and Leucocytozoon were 33.03%, 1.21%, and 0.6%, respectively. Sequences analysis has detected 43 lineages in Iranian birds’ hosts. Lineages were attributed to three genera Haemoproteus (n = 37), Plasmodium (n = 4), and Leucocytozoon (n = 2), of which 23 lineages fully matched previously recorded sequences in GenBank and MalAvi data reciprocities. Five lineages of ACDUM1, ACDUM2, PARUS1, PYERY01, and SISKIN1 were detected in multiple hosts’ species from dissimilar families. In Bayesian tree, all sequences were clustered in three main monophyletic clades as Haemoproteus, Plasmodium, and Leucocytozoon genera. Conclusions/Significance As the first study outlining the molecular detection of hematozoa of passerines from Iran, the current study has recorded 20 new lineages for three genera of Haemoproteus, Plasmodium, and Leucocytozoon. Additional investigations into these taxa in the avifauna for the other parts of Iran may provide extra information on blood parasites, hosts relationships and distribution patterns.

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