A referee analysis method for the detection and quantification of Pacific ciguatoxins in fish flesh has recently been established by the public health analytical laboratory for the State of Queensland, Australia. Fifty-six fish samples were analysed, which included 10 fillets purchased as negative controls. P-CTX-1 was identified in 27 samples, and P-CTX-2 and P-CTX-3 were found in 26 of those samples. The range of P-CTX-1 concentrations was 0.04-11.4 姯kg fish flesh; coefficient of variation from 90 replicate analyses was 7.4%. A liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) method utilising a rapid methanol extraction and clean-up is reliable and reproducible, with the detection limit at 0.03 姯kg fish flesh. Some matrix effects are evident, with fish oil content a likely signal suppression factor. Species identification of samples by DNA sequence analysis revealed some evidence of fish substitution or inadvertent misidentification, which may have implications for the management and prevention of ciguatera poisoning. Blinded inspection of case notes from suspect ciguatera poisoning cases showed that reporting of ciguatera-related paraesthesias was highly predictable for the presence of ciguatoxins in analysed fish, with 13 of 14 expected cases having consumed fish that contained P-CTX-1 (p < 0.001, Fishers Exact Test).