Stable carbon and nitrogen isotope ratios (δ13C and δ15N) of serum, red blood cells (RBC), muscle, and blubber were measured in captive and wild northeast Pacific harbor seals (Phoca vitulina richardii) at three coastal California sites (San Francisco Bay, Tomales Bay, and Channel Islands). Trophic discrimination factors (ΔTissue-Diet) were calculated for captive seals and then applied in wild counterparts in each habitat to estimate trophic position and feeding behavior. Trophic discrimination factors for δ15N of serum (+3.8‰), lipid-extracted muscle (+1.6‰), and lipid-blubber (+6.5‰) are proposed to determine trophic position. An offset between RBC and serum of +0.3‰ for δ13C and −0.6‰ for δ15N was observed, which is consistent with previous research. Specifically, weaner seals (<1 yr) had large offsets, suggesting strong trophic position shifts during this life stage. Isotopic values indicated an average trophic position of 3.6 at both San Francisco Bay and Tomales Bay and 4.2 at Channel Islands. Isotopic means were strongly dependent on age class and also suggested that mean diet composition varies considerably between all locations. Together, these data indicate that isotopic composition of blood fractions can be an effective approach to estimate trophic position and dietary behavior in wild pinnipeds.