Introduction: Plasmodium falciparum apical membrane antigen 1 (AMA1) is a blood stage protein involved with erythrocyte invasion. Because anti-AMA1 antibodies inhibit parasite growth and are associated with clinical immunity, AMA1 is a leading malaria vaccine candidate in clinical testing. However, AMA1 is highly polymorphic and antibodies elicited by one variant of AMA1 may not confer protection against others; therefore, an effective vaccine might need to include multiple variants. Studies of antibody cross-reactivity to AMA1 variants would aid in the rational design of an AMA1 vaccine. The current study used an autotransporter expression system to express several variant AMA1 fragments. We expected that antibodies raised against one P. falciparum AMA1 variant would recognize homologous AMA1 fragments but not highly dissimilar variants. Methods: Fragments of the AMA1 protein corresponding to AMA1 sequences from six P. falciparum strains (3D7, FVO, DD2, 7G8, Fab9, and M5) were genetically engineered and incorporated into an EspP autotransporter. Proteins were expressed, purified and used in western blotting assays to assess reactivity of polyclonal rabbit antibodies that were raised against recombinant 3D7-AMA1 and FVO-AMA1. Results: Anti-3D7 AMA1 polyclonal rabbit antibodies preferentially recognized the 3D7 AMA1 fragments, and cross-reacted with the heterologous Fab9 AMA1 fragments via western blotting. Polyclonal rabbit antibodies raised against FVO AMA1 preferentially recognized FVO AMA1 fragments but also had a higher level of cross-reactivity, strongly recognizing heterologous M5, and 7G8 AMA1 fragments. Conclusions: The autotransporter expression system allowed for characterization of polypeptide fragments from different AMA1 strains based on antibody recognition. It was evident that antibody recognition became more specific when larger fragments were studied. The identification of residues that form the basis of cross-reactivity between strains could yield useful information to guide the design of future AMA1 vaccines.