Antimicrobial peptides (AMPs) are a major part of the innate immune defence system which shows a broad spectrum of activity, defending the host against invading microbes. The aim of this work was to identify the AMPs present in yellowtail kingfish (Seriola lalandi) and pouched lamprey (Geotria australis) and use molecular techniques to fully sequence their cDNA and quantify their expression in adult individuals. Using bioinformatic approaches candidate AMP genes were ascertained from available S. lalandi and G. australis RNA-seq transcriptomic databases, obtained from various tissues. Selected AMPs were chosen to have their full cDNA sequence amplified using RACE-PCR, which were then cloned and sequenced. Complete cDNA sequences were obtained for S. lalandi hepcidin and moronecidin, whereas attempts to complete the G. australis defensin-like cDNA were unsuccessful. Comparison of the S. lalandi hepcidin and moronecidin protein sequences with proteins already characterised in other fish showed good homology and conservation of important features. In addition, specific primers were designed to examine the expression levels of S. lalandi hepcidin and moronecidin in gill, liver or spleens of three fish. Analysis showed hepcidin expression to be highest in liver tissues, whereas moronecidin expression was highest in the gills and spleens. This study provides a comprehensive overview of the AMP genes present in S. lalandi and G. australis and some initial characterisation of S. lalandi hepcidin and moronecidin, which will permit the development of future research applications. Overall, characterising AMP genes in jawed and jawless vertebrates is vital for economical and successful fish farming, while also providing possible therapeutic benefits associated with AMP research in biomedicine and disease in wild fish stocks.