Mutagenesis-based screens in mice are a powerful discovery platform to identify novel genes or gene functions associated with disease phenotypes. An N-ethyl-N-nitrosourea (ENU) mutagenesis screen induces single nucleotide variants randomly in the mouse genome. Subsequent phenotyping of mutant and wildtype mice enables the identification of mutated pathways resulting in phenotypes associated with a particular ENU lesion. This unbiased approach to gene discovery conducts the phenotyping with no prior knowledge of the functional mutations. Before the advent of affordable next generation sequencing (NGS), ENU variant identification was a limiting step in gene characterization, akin to ‘finding a needle in a haystack’. The emergence of a reliable reference genome alongside advances in NGS has propelled ENU mutation discovery from an arduous, time-consuming exercise to an effective and rapid form of mutation discovery. This has permitted large mouse facilities worldwide to use ENU for novel mutation discovery in a high-throughput manner, helping to accelerate basic science at the mechanistic level. Here, we describe three different strategies used to identify ENU variants from NGS data and some of the subsequent steps for mutation characterisation.