In the present study, we applied a novel high-throughput in vitro gastrointestinal digestion model to phenotype bioaccessibility of phenolics in a diverse germplasm collection representing cultivated highbush blueberries. Results revealed significant (P < 0.05) differences between accessions, years, and accession by year interaction for relative and absolute bioaccessibility of flavonoids and phenolic acids. Broad sense heritability estimates revealed low to moderate inheritances of relative and absolute bioaccessibility, suggesting that besides environmental variables, genetics factors could control bioaccessibility of phenolics. Acylated anthocyanins had significantly higher relative bioaccessibility than non-acylated anthocyanins. Correlation analysis indicated that relative bioaccessibility did not show significant association with fruit quality or raw concentration of metabolites. The study also identified accessions that have high relative and absolute bioaccessibility values. Overall, combining the bioaccessibility of phenolics with genetic and genomic approaches will enable the identification of genotypes and genetic factors influencing these traits in blueberry.