Developing efficient, reliable, cost‐effective ways to identify diet is required to understand trophic ecology in complex ecosystems and improve food web models. A combination of techniques, each varying in their ability to provide robust, spatially and temporally explicit information can be applied to clarify diet data for ecological research. This study applied an integrative analysis of a fishery‐targeted species group—Plectropomus spp. in the central Great Barrier Reef, Australia, by comparing three diet‐identification approaches. Visual stomach content analysis provided poor identification with ~14% of stomachs sampled resulting in identification to family or lower. A molecular approach was successful with prey from ~80% of stomachs identified to genus or species, often with several unique prey in a stomach. Stable isotope mixing models utilizing experimentally derived assimilation data, identified similar prey as the molecular technique but at broader temporal scales, particularly when prior diet information was incorporated. Overall, Caesionidae and Pomacentridae were the most abundant prey families (>50% prey contribution) for all Plectropomus spp., highlighting the importance of planktivorous prey. Less abundant prey categories differed among species/color phases indicating possible niche segregation. This study is one of the first to demonstrate the extent of taxonomic resolution provided by molecular techniques, and, like other studies, illustrates that temporal investigations of dietary patterns are more accessible in combination with stable isotopes. The consumption of mainly planktivorous prey within this species group has important implications within coral reef food webs and provides cautionary information regarding the effects that changing resources could have in reef ecosystems.

Tropical reefs are highly diverse ecosystems, and reliable biomonitoring, through diet metabarcoding, is needed to understand present and future trophic relationships in this changing habitat. Several studies have assessed the reliability and effectiveness of single molecular markers; however, a cross-marker validation has rarely been performed. This study identified crucial properties for 12S rDNA, 16S rDNA and COI metabarcoding in tropical-reef piscivores (Plectropomus spp.). In addition, three new versatile primer sets for 16S were designed in silico for metabarcoding of reef fish. Results showed that COI was overall better at recovering true diversity because of a well-supported database. Second, optimal 16S amplicon sizes ranged between 160 and 440 base pairs for full diversity recovery, with increased species detection for the 270-base pairs region. Finally, blocking of predator-specific COI sequences was not equally effective in all host species, potentially introducing bias when diet compositions are directly compared. In conclusion, this novel study showed that marker success for prey identification is highly dependent on the reference database, taxonomic scope, DNA quality, amplicon length and sequencing platform. Results suggest that COI, complemented with 16S, yields the best outcome for diet metabarcoding in reef piscivores. Findings in this paper are relevant to other piscivores and other metabarcoding applications.