Zebrafish have become an invaluable model for studying blood and immune cell development and function, with strong conservation with humans at the cell and gene levels. However, the natural killer (NK) cell population has been understudied in zebrafish. This project seeks to address this knowledge gap by developing markers to enable the identification, isolation, and characterization of zebrafish NK cells to investigate their biology and role in disease. A number of candidate genes were chosen as potential markers based on published RNAseq data. Whole mount in site hybridisation with anti-sense RNA probes identified two NK lysin genes, nkl.2 and nkl.4, and one encoding the transcription factor zbtb32 that were specifically expressed from 5 days post fertilisation (dpf) in the thymus, the initial site of lymphoid cell development. In contrast, the non-specific cytotoxic cell marker, nccrp1, showed expression in the digestive tract from 3 dpf, which shifted to the thymus at 7 dpf. Expression was also assessed by RT-PCR in zebrafish harbouring mutations in components of the key pathway regulating mammalian NK production or in response to specific immune modulators. Significantly decreased expression of all genes was observed in knockout mutants of IL-2 receptor gamma common (il2rgc), while in hyperactive mutants of the downstream Janus kinase 3 (jak3) increased expression was observed in all genes except nccrp1. A marked increase in expression of nkl.2 and nkl.4 occurred following injection of polyinosinic:polycytidylic acid (poly(I:C)), a mimic of viral infection known to activate NK cells, but not with lipopolysaccharide (LPS), a mimic of bacterial infection. Together, these observations suggest nkl.2 and nkl.4 might serve as useful markers of zebrafish NK cells to support ongoing research on NK biology and role in disease, including for therapeutic innovations.