This protocol describes the design of primer pairs against the human genome for the synthesis of probes for high-definition DNA FISH (HD-FISH). This pipeline selects PCR primer pairs with optimal thermodynamic features, delimiting amplicons 200\u2013220 nucleotides in length, and filters out primer pairs amplifying multiple targets as well as cross-hybridizing amplicons. Using such primers, highly specific double-stranded probes can be rapidly generated for virtually any desired genomic locus by fluorescently labeling pooled amplicons after PCR.While this protocol describes the design against the human genome, we have also used it to geneate a genome-wide library for mouse. The design and method should work across other organisms as well.For more information please see the full paper (and the dedicated hdfish.eu website):Bienko, Magda et al. \u201cA Versatile Genome-Scale PCR-Based Pipeline for High-Definition DNA FISH.\u201d Nature methods 10.2 (2013): 122\u2013124. PMC. Web. 9 Nov. 2015.