In this study, we evaluated the impact of hemoglobin (Hb) variants on the performance of the Abbott Alinity c and Bio-Rad Variant II Turbo 2.0 HPLC Hb A1c assays. The analytical performance of the Abbott Alinity c Hb A1c (enzymatic) assay was compared to the Bio-Rad Variant II Turbo 2.0 HPLC method using leftover whole blood EDTA samples with and without the presence of a hemoglobin variant. Assay precision was determined from an analysis of controls. Bias was estimated from analysis of a set of 40 samples analyzed by a Tosoh G8 HPLC instrument at the University of Missouri Diabetes Diagnostic Laboratory, an NGSP Secondary Reference Laboratory. Between-day precision was excellent for both methods (<3%). Bias met NGSP criteria of ±5% to target value. Correlation between the Alinity and Bio-Rad methods was good for patient samples without a hemoglobinopathy (y = 1.028x - 0.38, standard error of the estimate (SEE) = 0.16, n = 36, mean bias = -0.22). A total of 700 hemoglobin variant samples were evaluated on the 2 methods. Of the hemoglobin variants, 640/700 gave results on both methods: hemoglobin (Hb) S trait (n = 452), C trait (n = 131), D trait (n = 23), E trait (n = 26), and a mixture of other hemoglobinopathies (n = 8) including beta thalassemia, high hemoglobin F, transfused Hb SC, transfused Hb SD, and transfused Hb SS, or unknown variant. There was good agreement for the 640 Hb variants between the methods with a range of mean differences of -0.10 to +0.06 depending on the variant, but more variability (SEE 0.25 to 0.39). Sixty samples did not have paired results. To our knowledge, this study was the largest investigation of the effect of hemoglobinopathies on the Abbott Alinity c Hb A1c assay. Analytical performance varied depending on the specific hemoglobin variant trait when compared to the Bio-Rad Variant II Turbo 2.0 HPLC method.