Author Summary Availability of bacterial “pan genomes,” based on multiple genome sequences of a given species, has revealed the existence of core genomes, but also of high levels of variable, nonconserved regions. The nature of bacterial strategies that assure genome organization while permitting biodiversity remains an intriguing question. A first clue in addressing this question comes from growing evidence for the existence of noncoding functional DNA motifs with specific distribution properties along the chromosome, which are implicated in DNA integrity. In this work, we addressed the challenging problem of predicting such motifs from pan-genome information. We analyzed characteristics of the “crossover hotspot instigator” motif, Chi, which, in E. coli, is an 8-base-pair sequence involved in chromosome maintenance. Our results show that Chi has specific distribution properties that are restricted to core genomes common to related E. coli strains. Using statistical modeling combined with comparative genomics, we then predicted the identity of Chi in core genomes of S. aureus and several streptococci, and confirmed them in vivo. The strategy developed in this study may be extended to reveal and characterize novel functional motifs, and should be instrumental in analyzing genome biodiversity mechanisms.