Increased recognition of the prevalence of human babesiosis in the United States, together with rising concern about the potential for transmission of this infection by blood transfusion, has provided motivation to develop definitive serologic and molecular tests for the causative agent, Babesia microti. To develop more sensitive and specific assays for B. microti, we screened a genomic expression library with patient serum pools. This screening resulted in the identification of three classes of novel genes and an additional two novel, unrelated genes, which together encode a total of 17 unique B. microti antigens. The first class (BMN1-2 family) of genes encodes seven closely related antigens with a degenerate six-amino-acid repeat that shows limited homology to Plasmodium sp. merozoite and sporozoite surface antigens. A second class (BMN1-8 family) of genes encodes six related antigens, and the third class (BMN1-17 family) of genes encodes two related antigens. The two remaining genes code for novel and unrelated sequences. Among the three classes of antigens and remaining novel sequences, five were chosen to code for the most immunodominant antigens (BMN1-2, -9, -15, and -17 and MN-10). Western blot analysis with the resulting recombinant proteins indicated that these antigens were targets of humoral immune responses during B. microti infection in humans.