Liver tumours in flatfish have been diagnosed using histopathology for decades in order to monitor the impacts of marine pollution in coastal and estuarine environments. This traditional method has been coupled with molecular analyses of tumours in the liver of the dab, Limanda limanda, in order to elucidate underpinning molecular level aetiology of such disease. A laser capture microdissection technique for molecular investigation of cancer has been applied in fish. The present study provides optimized steps for environmental sample utilisation: a procedure for field sample collection and handling; a method allowing reliable identification of lesions on frozen tissue sections; and, downstream molecular analyses of tumourigenesis markers (retinoblastoma gene) in laser microdissected samples. This approach facilitates the use of wild flatfish as a model of environmentally-induced tumourigenesis, and has wide applicability to any disease pathology for which the underpinning molecular aetiology is required.