Reverse genetics in the zebrafish has been neglected because of the lack of an efficient gene targeting technology. A project to sequence and finish the zebrafish genome has contributed to the effectiveness of a process termed TILLING, which can be used to create targeted alleles in genes of choice by re-sequencing exons across libraries of mutagenised individuals. We have previously used this technique to generate a large library of over 500 zebrafish non-sense alleles using PCR amplification of 4224 exons selected from genes requested by the zebrafish research community. With the increase of sequencing throughput from next generation sequencing technologies, it has become feasible to consider re-sequencing entire exomes from mutagenised individuals. Using the Agilent Sureselect system, a whole exome enrichment reagent has been designed using the gene build from the Zv8 zebrafish genome assembly. Whole exomes from mutagenised individuals have been enriched and re-sequenced on the Illumina platform. Potential non-sense and essential splice mutations have been identified and confirmed by Kasp genotyping (allele specific amplification). To date 381 mutagenised individuals have been exome sequenced, and 4163 functionally relevant alleles in 3535 genes have been identified (over 13% of all protein coding genes). F2 families, generated by outcrossing the mutagenised individuals, have been genotyped to identify carriers of the mutations that were successfully confirmed. Embryos from F3 incrosses have been phenotypically characterized during the first 5 days of embryonic development, and we have shown that we are able to successfully link morphological phenotypes to genotypes from the sequencing data. Using the methods described here, it is possible to efficiently identify KO alleles in the Zebrafish and we plan to sequence 10,000 individual exomes over the next 5 years. Indeed, with this whole exome sequencing approach, we hope to identify a non-sense allele in every protein coding gene in the zebrafish.