Variation in antioxidant activity (AA), total phenolic content (TPH), and total anthocyanin content (ACY) was examined in 1998 and 1999 in fruit of 52 (49 blue-fruited and 3 pink-fruited) genotypes from a blueberry breeding population. The species ancestry included Vaccinium corymbosum L. (northern highbush blueberry), V. angustifolium Ait. (lowbush blueberry), V. constablaei Gray (mountain highbush blueberry), V. ashei Reade (rabbiteye blueberry), and V. myrtilloides Michx. (lowbush blueberry). Using a methyl linoleate oxidation assay (MeLO) on acidified methanolic extracts of the berries, a 5-fold variation was found in AA in 1998 and a 3-fold variation in 1999 among the blue-fruited genotypes. Analyses of variance (ANOVA) revealed variation among genotypes (P < 0.0001) in single and combined years, regardless of inclusion of pink-fruited selections and adjustment for berry size. While mean AA of all genotypes did not change between the 2 years, ranking of some genotypes for AA changed significantly between 1998 and 1999. Of the 10 genotypes that demonstrated the highest AA in 1998, four were among the 10 genotypes that demonstrated highest AA in 1999. Similarly, of the 15 genotypes with the highest AA, 10 were the same both years. As with AA, mean TPH of all genotypes did not change between years and ANOVA demonstrated genotypic variation regardless of adjustment for berry size/weight or exclusion of pink-fruited selections. Changes in genotype rank occurred between years. The difference in TPH between lowest- and highest-ranking blue-fruited genotypes was ≈2.6-fold in both 1998 and 1999. Seven of the 10 highest-ranking genotypes were the same both years and TPH correlated with AA (r = 0.92, P < 0.01) on a genotype mean basis for combined years. ACY correlated less well with AA (r = 0.73, P < 0.01 for combined years). When genotypes were categorized into six groups according to species ancestry, V. myrtilloides and V. constablaei × V. ashei crosses ranked highest and second highest, respectively, for AA in both years. The groups comprised of V. corymbosum genotypes, V. angustifolium genotypes, and those with both V. corymbosum and V. angustifolium in their lineage were indistinguishable from each other. Samples from some of the genotypes were analyzed for oxygen radical absorbance capacity and ferric-reducing antioxidant power, and these aqueous-based antioxidant assays correlated well with the lipid emulsion-based MeLO (all r ≥ 0.90, P < 0.01). The three antioxidant assays may be equally useful for screening in a blueberry breeding program and the choice of assay may depend on the goal of the program and the resources available.