Hemoplasma DNA was found in 22% (12/55) of samples from six bat species in Chile, including one of three big-eared brown bats (Histiotus macrotus), one of three small big-eared brown bats (Histiotus montanus), and 10 of 27 Chilean myotis (Myotis chiloensis). Characterization of the 16S ribosomal RNA gene identified three genotypes phylogenetically related to other bat, rodent, and human hemoplasmas.