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Muscle melanisation in sand flathead is defined as the presence of abnormal black spots in the fish's flesh and the phenomenon has been associated with Zn pollution in the environment. In this study, Zn levels in the fish muscle and crabs were analysed to investigate the role of Zn in causing muscle melanisation and the fish's uptake of Zn via its diet, crabs. An improved technique for extracting melanised fish muscle from fillets with black spots was developed. It enabled Zn levels in melanised muscle to be more accurately determined when compared to previous study and showed Zn levels were 2.1–3.5 times higher in melanised regions of muscle than non-melanised regions of muscle from fish with melanisation and fish unaffected by melanisation. This indicated elevated levels of Zn were localised in the melanised muscle. Muscle melanisation was potentially caused by Zn ions binding to the active site of tyrosinase, an enzyme involved in melanin production, likely facilitated by the presence of higher Zn than Cu in the polluted estuaries. A digestion reagent mixture suitable for microwave assisted acid digestion and analysis of Zn in the whole crab was developed using a mixture of HNO3 and H2O2. Zn levels in the whole crab were 1.5 times higher in 50% of the crab species studied from a polluted estuary compared to the same species from an unpolluted estuary. As sand flathead primarily feed on crabs, the fish's diet was a likely source of elevated Zn for the fish. Overall, this study has described new techniques useful for the study of melanised fish and heavy metal levels in the whole crab. The findings from this study also provided insights that will inform and guide the direction of future research on muscle melanisation in sand flathead.