With the recent advent of genomic tools for cattle, several mutations affecting fertility have been identified and selected against, such as AM, PHA, DUMPS, CVM and BLAD. In an attempt to identify unknown recessive lethal alleles in the current dairy population, a search using deep Mendelian sampling of more than 100,000 Holstein cattle was conducted for high-frequency haplotypes that have a deficit of homozygotes at the population level. This search led to the discovery of a putative recessive lethal in Holstein dairy cattle on BTA5. The haplotype denoted HH1 was associated with reduced fertility, and further investigation identified one highly-influential Holstein bull as the source ancestor. By combining whole-genome resequencing, high-density genotyping and haplotyping techniques, a nonsense mutation in APAF1 was identified, which is predicted to truncate approximately one-third of the encoded APAF1 protein. No homozygous recessive individuals were found in 758 genotyped animals, whereas all known carriers and carrier haplotypes possessed one copy of the mutant allele. Functional annotation of APAF1 supports our findings and the gene’s vital role in development. This newly identified lethal has been responsible for a substantial number of spontaneous abortions in Holstein dairy cattle throughout the past half-century. With the mutation identified, selection against the deleterious allele in breeding schemes will aid in eliminating this defect from the population. Whole-genome resequencing thus proved to be a powerful strategy to rapidly identify a deleterious mapped mutation in a known carrier of a recessive lethal allele.