Directional genomic hybridization (dGH) is a strand-specific fluorescence in situ hybridization (FISH) technology developed for the detection of historically difficult to detect chromosomal inversions and related cryptic structural variants. The methodology employs incorporation of halogenated nucleotide analogs during a single round of DNA replication, followed by selective photolysis and exonuclease digestion to generate single-stranded chromatids representing parental DNA strands of opposite orientation. Hybridization with fluorescently tagged, bioinformatically designed single-stranded oligonucleotide probe sets to unique sequences of defined 5′-to-3′ orientation results in labelling of only one chromatid. Thus, intrachromosomal inversions appear as distinct signal “switches” from one sister chromatid to the other. Since its initial conception, dGH has evolved from an academic discovery tool into a versatile assay with a variety of applications, ranging from evolutionary genomics, radiation biodosimetry, and clinical cytogenetics, to cancer benchmarking, telomere biology, and most recently development and regulatory assessment of genome-edited therapeutic products.