De novo germline mutations arise preferentially in male owing to fundamental differences between spermatogenesis and oogenesis. Post-meiotic chromatin remodeling in spermatids results in the elimination of most of the nucleosomal supercoiling and is characterized by transient DNA fragmentation. Using three alternative methods, DNA from sorted populations of mouse spermatids was used to confirm that double-strand breaks (DSB) are created in elongating spermatids and repaired at later steps. Specific capture of DSB was used for whole-genome mapping of DSB hotspots (breakome) for each population of differentiating spermatids. Hotspots are observed preferentially within introns and repeated sequences hence are more prevalent in the Y chromosome. When hotspots arise within genes, those involved in neurodevelopmental pathways become preferentially targeted reaching a high level of significance. Given the non-templated DNA repair in haploid spermatids, transient DSBs formation may, therefore, represent an important component of the male mutation bias and the etiology of neurological disorders, adding to the genetic variation provided by meiosis.