The zebrafish mutation project (ZMP) aims to generate a loss of function allele for every protein-coding gene, but importantly to also characterise the phenotypes of these alleles during the first five days of development. Such a large-scale screen requires a systematic approach both to identifying phenotypes, and also to linking those phenotypes to specific mutations. This phenotyping pipeline simultaneously assesses the consequences of multiple alleles in a two-step process. First, mutations that do not produce a visible phenotype during the first five days of development are identified, while a second round of phenotyping focuses on detailed analysis of those alleles that are suspected to cause a phenotype. Allele-specific PCR single nucleotide polymorphism (SNP) assays are used to genotype F2 parents and individual F3 fry for mutations known to be present in the F1 founder. With this method specific phenotypes can be linked to induced mutations. In addition a method is described for cryopreserving sperm samples of mutagenised males and their subsequent use for in vitro fertilisation to generate F2 families for phenotyping. Ultimately this approach will lead to the functional annotation of the zebrafish genome, which will deepen our understanding of gene function in development and disease.