The hemoparasite Babesia can cause life-threatening infections to neonates, elderly and immunocompromised people, and people who have undergone splenectomy. By using pooled hamster serum samples collected 21 days after infection with Babesia microti, we developed an immunohistochemical assay for formalin-fixed, paraffin-embedded tissue (FFPET) samples and blood smears. By use of the immunohistochemical assay, parasites were detected inside erythrocytes present in the heart, spleen, and liver of experimentally and naturally infected animals. FFPET samples from 2 fatal and 1 nonfatal human cases demonstrated immunohistochemical assay-positive parasites in circulating erythrocytes in various organs, including lymph nodes and spleen. In addition, air-dried blood smears from 4 patients showed positive immunohistochemical staining inside the erythrocytes. The immunohistochemical assay showed cross-reactivity against the Babesia WA-1 strain but did not react against Babesia bigemina or Plasmodium falciparum. The immunohistochemical assay for Babesia microti successfully detected parasites in human and animal FFPET samples and blood smears. This technique will be useful for the diagnosis of clinically suspected cases and for differentiating Babesia microti infection from malaria. Application of this technique to animal models will better define pathogenic mechanisms, including the possible recognition of exoerythrocytic tissue stages.