Plasmodiumspp. and some other blood parasites belonging to the order Haemosporida are the focus of many epidemiological studies worldwide. However, haemosporidian parasites from wild animals are largely neglected in scientific research. For example,Polychromophilusparasites, which are exclusive to bats, are described in Europe, Asia, Africa, and Oceania, but little is known about their presence and genetic diversity in the New World. In this study, 224 samples of bats from remaining fragments of the Atlantic Forest and Pantanal biomes, as well as urbanized areas in southern and southeastern Brazil, were analyzed for the presence of haemosporidian parasites by PCR of the mitochondrial gene that encodes cytochrome b (cytb). The PCR fragments of the positive samples were sequenced and analyzed by the Bayesian inference method to reconstruct the phylogenetic relationships betweenPolychromophilusparasites from bats in Brazil and other countries. Sequences from Brazilian lineages ofPolychromophiluswere recovered in a clade with sequences fromPolychromophilus murinusand close to the onePolychromophilussequence obtained in Panama, the only available sequence for the American continent. This clade was restricted to bats of the family Vespertilionidae and distinct fromPolychromophilus melanipherus, a parasite species mainly found in bats of the family Miniopteridae. The detection ofPolychromophilusand the genetic proximity toP. murinuswere further confirmed with the amplification of two other genes (clpcandasl). We also found a Haemosporida parasite sequence in a sample ofNoctilio albiventriscollected in the Pantanal biome, which presents phylogenetic proximity with avianHaemoproteussequences. Morphological and molecular studies are still needed to conclude and describe thePolychromophilusspecies in BrazilianMyotisbats in more detail and to confirmHaemoproteusparasites in bats. Nevertheless, these molecular results in Brazilian bats confirm the importance of studying these neglected genera.