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Strawberries ( Fragaria ×ananassa ) are highly sought-after fruit because of their flavor, color, and nutritional benefits. The balance of the soluble solids content (SSC) and titratable acidity (Tacid) contribute to the overall strawberry taste and perceived sweetness, while the total monomeric anthocyanin content (TMAC) is the primary basis for fruit color. Although North Carolina has an active strawberry breeding program to develop genotypes adapted to the South Atlantic region of the United States, parental germplasm has not been characterized. In this study, a greenhouse collection of fruit from 268 commercial cultivars and advanced breeding lines was used to characterize fruit composition diversity. Strawberry genotypes (all octoploids) were separated into four distinct clusters based on fruit composition profiles and multivariate analysis results. Genotypes in cluster 1 exhibited the lowest SSC (7.0%), Tacid (0.72%), and TMAC (31.22 mg/100 g), whereas cluster 2 had the second lowest %SSC and %Tacid and highest TMAC (54.57 mg/100 g). Cluster 3 was characterized by high SSC (10.3%) and pH (3.66), and fruit from cluster 4 had the highest average Tacid (1.21%) and lowest pH (3.43). Genotypes from Florida were found in the same cluster, while California genotypes were split among clusters 1 and 2. The first principal components (PCs; PC1 and PC2) accounted for 64.88% of the total variance within total fruit composition, with pH and Tacid contributing to PC1 (91.1%), SSC contributing to PC2 (71.1%), and TMAC associated with PC3 (77.4%). These differences in fruit composition among genotypes in the North Carolina core germplasm collection will be useful in the determination of future crosses in the breeding program.