Spinach leaves harvested at three maturity stages from eight commercial cultivars (CC) and eight advanced breeding lines (ABL) were evaluated for oxygen radical absorbing capacity (ORAC), total phenolics, and flavonoid composition and content. ABL had higher levels of total phenolics, total flavonoids, and ORAC than CC. Midmaturity spinach leaves had higher levels of total phenolics, total flavonoids, and antioxidant capacity than immature and mature leaves. The contents of individual flavonoids varied in response to maturation, with the predominant glucuronated flavones decreasing and patuletin and spinacetin derivatives increasing. Both total phenolics and total flavonoids correlated well with ORAC (r(xy)() = 0.78 and 0.81, respectively) demonstrating that flavonoids were major contributors to antioxidant capacity. Our results indicate that spinach genotypes should be harvested at the midmaturity stage for consumers to benefit from elevated levels of health promoting flavonoids present in the leaves. Additionally, plant breeders can select for increased phenolic content to increase antioxidant capacity of spinach genotypes.