DNA metabarcoding is used to enumerate and identify taxa in both environmental samples and tissue mixtures. The composition and resolution of metabarcoding data depend on the primer(s) used. Markers that amplify different genes can mitigate biases in primer affinity, amplification efficiency, and reference database resolution, but few empirical studies have evaluated markers for complementary performance. Here, we assess the individual and joint performance of 22 markers for detecting species in a DNA pool of >100 species of primarily marine and freshwater fishes, but also including representatives of elasmobranchs, cephalopods, and crustaceans. Marker performance includes the integrated effect of primer specificity and reference availability. We find that a portfolio of four markers targeting 12S, 16S, and multiple regions of COI identifies 100% of reference taxa to family and nearly 60% to species. We then use the four markers in this portfolio to evaluate metabarcoding of heterogeneous tissue mixtures, using experimental fishmeal to test: 1) the tissue input threshold to ensure detection; 2) how read depth scales with tissue abundance; and 3) the effect of non-target material in the mixture on recovery of target taxa. We consistently detect taxa that make up >1% of fishmeal mixtures and can detect taxa at the lowest input level of 0.01%, but rare taxa (<1%) were detected inconsistently across markers and replicates. Read counts showed weak correlation with tissue input, suggesting they are not a valid proxy for relative abundance. Despite this limitation, our results demonstrate the value of a primer portfolio approach—tailored to the taxa of interest—for detecting and identifying both rare and abundant species in heterogeneous tissue mixtures.