Studying genetic variations in the human genome is important for understanding phenotypes and complex traits, including rare personal variations and their associations with disease. The interpretation of polymorphisms requires reliable methods to isolate natural genetic variations, including combinations of variations, in a format suitable for downstream analysis. Here, we describe a strategy for targeted isolation of large regions (∼35 kb) from human genomes that is also applicable to any genome of interest. The method relies on recombineering to fish out target fosmid clones from pools and thereby circumvents the laborious need to plate and screen thousands of individual clones. To optimize the method, a new highly recombineering-efficient bacterial host, including inducible TrfA for fosmid copy number amplification, was developed. Various regions were isolated from human embryonic stem cell lines and a personal genome, including highly repetitive and duplicated ones. The maternal and paternal alleles at the MECP2/IRAK 1 loci were distinguished based on identification of novel allele-specific single-nucleotide polymorphisms in regulatory regions. Additionally, we applied further recombineering to construct isogenic targeting vectors for patient-specific applications. These methods will facilitate work to understand the linkage between personal variations and disease propensity, as well as possibilities for personal genome surgery.