To identify novel genomic regions that regulate sex determination, we utilize the powerful C57BL/6J-Y(POS) (B6-Y(POS)) model of XY sex reversal where mice with autosomes from the B6 strain and a Y-chromosome from a wild-derived strain, Mus domesticus poschiavinus (Y(POS)), show complete sex reversal. In B6-Y(POS), the presence of a 55-Megabase (Mb) congenic region on chromosome 11 protects from B6-Y(POS) sex reversal in a dose dependent manner. Using mouse genetics backcross designs and high-density single nucleotide polymorphism (SNP) arrays, we narrowed the congenic region to a 1.5 Mb genomic on chromosome 11 that confers 80% protection from B6-Y(POS) sex reversal when one copy is present and complete protection when two copies are present. It was previously believed that the protective congenic region originated from the 129S1/SviMJ (129) strain. However, genomic analysis revealed that this region is not derived from 129 and most likely is derived from the semi-inbred strain POSA. We show that the small 1.62 Mb congenic region is located within the Sox9 promoter protects against B6-Y(POS) sex reversal and promotes the expression of Sox9, thereby driving testis development within the B6-Y(POS) background. Through 30 years of backcrossing, this congenic region was maintained, as it promoted male sex determination and fertility despite the female-promoting B6-Y(POS) genetic background. Our findings demonstrate that long-range enhancer regions are critical to developmental processes and can be used to identify the complex interplay between genome variants, epigenetics, and developmental gene regulation.