The regions of the paternal genome susceptible to oxidative stress have not been elucidated. However, oxidative DNA damage in spermatozoa can influence the incidence of de novo mutations in children due to defective repair in the oocyte prior to the first mitotic division.. Current approaches to extract and fragment DNA from mammalian spermatozoa provide several challenges into investigating oxidative damage carried in the genome of male gametes. Reducing agents, like Dithiothreitol (DTT) and Beta-Mercaptoethanol (β-ME) induce oxidative DNA damage. DNA shearing techniques used in the preparation of samples for immunoprecipitation and next-generation sequence also introduce cofounding agents that reduce the accuracy of results obtained. Using a modified DNA immunoprecipitation approach, we adapted and optimised methodologies that minimise or completely removed exposure to DNA damaging compounds from the standard extraction and fragmentation procedures. Our modified DNA immunoprecipitation was used to isolate oxidised DNA from oxidatively-stressed human spermatozoa, followed by genome-wide sequencing and bioinformatic analyses. This strategy identified ~9,000 regions highly vulnerable to oxidative damage and these varied in size from 150bp to 1000bp. Specific chromosomes showed differential susceptibility to damage, chromosome 15 was particularly vulnerable while the sex chromosomes were protected. Susceptible regions generally lay outside protamine- and histone-packaged domains, and strongly associated with SINEs and LINEs, centromeres, and telomeres. Vulnerable domains were confirmed to be highly oxidised in infertile patients. Identification of genomic regions susceptible to oxidation in the male germ line represents an important step in understanding the implications of oxidative DNA damage for fertility and offspring development.