Malaria caused by Plasmodium falciparum is a major cause of global infant mortality, and no effective vaccine currently exists. Multiple potential vaccine targets have been identified, and immunoepidemiology studies have played a major part in assessing those candidates. When such studies are carried out in high-transmission settings, individuals are often superinfected with complex mixtures of genetically distinct P. falciparum types, making it impossible to directly correlate the genotype of the infecting antigen with the antibody response. In contrast, in regions of low transmission P. falciparum infections are often genetically simple, and direct comparison of infecting genotype and antigen-specific immune responses is possible. As a test of the utility of this approach, responses against several domains and allelic variants of the vaccine candidate P. falciparum merozoite surface protein 3 (PfMSP3) were tested in serum samples collected near Iquitos, Peru. Antibodies recognizing both the conserved C-terminal and the more variable N-terminal domain were identified, but anti-N-terminal responses were more prevalent, of higher titers, and primarily of cytophilic subclasses. Comparing antibody responses to different PfMSP3 variants with the PfMSP3 genotype present at the time of infection showed that anti-N-terminal responses were largely allele class specific, but there was some evidence for responses that cross-reacted across allele classes. Evidence for cross-reactive responses was much stronger when variants within one allele class were tested, which has implications for the rational development of genotype-transcending PfMSP3-based vaccines. © 2011, American Society for Microbiology.