Antigen polymorphisms in essential malarial antigens are a key challenge to the design and development of broadly effective malaria vaccines. The effect of polymorphisms on antibody responses is fairly well studied while much fewer studies have assessed this for T cell responses. This study investigated the effect of allelic polymorphisms in the malarial antigen apical membrane antigen 1 (AMA1) on ex vivo T cell-specific IFN-γ responses in subjects with lifelong exposure to malaria. Human leukocyte antigen (HLA) class I-restricted peptides from the 3D7 clone AMA1 were bioinformatically predicted and those with variant amino acid positions used to select corresponding allelic sequences from the 7G8, FVO, FC27 and tm284 parasite strains. A total of 91 AMA1 9-10mer peptides from the five parasite strains were identified, synthesized, grouped into 42 allele sets and used to stimulate PBMCs from seven HLA class 1-typed subjects in IFN-γ ELISpot assays. PBMCs from four of the seven subjects (57%) made positive responses to 18 peptides within 12 allele sets. Fifty percent of the 18 positive peptides were from the 3D7 parasite variant. Amino acid substitutions that were associated with IFN-γ response abrogation were more frequently found at positions 1 and 6 of the tested peptides, but substitutions did not show a clear pattern of association with response abrogation. Thus, while we show some evidence of polymorphisms affecting T cell response induction, other factors including TCR recognition of HLA-peptide complexes may also be at play.