Hemolytic Disease of the Fetus and Newborn (HDFN) is a genetic disorder caused by blood group incompatibility, particularly Rh (D) antigen, in the parental couple. This study aimed to determine the prevalence of blood donor women exposed to the risk of HDFN among 913 blood donors in the southern region of Benin. The study was conducted from March to June 2022 at the departmental blood transfusion centers in the Atlantic and Littoral regions. Samples were collected following the procedures of the National Blood Transfusion Agency (NBTA), using tube and microplate agglutination tests. Among the donors, 260 (28.5%) were women aged between 18 and 52 years. The O+ blood group donors constituted the largest proportion, accounting for 42% of the sample. Nearly one-third (33.3%) of the female blood donors were Rh (D) negative, putting them at risk of HDFN. Most of these women were of reproductive age, with a significant representation in the 20-30 age groups (42.1%). This study revealed that one-third of female blood donors in the Atlantic and Littoral regions were at risk of giving birth to an infant affected by HDFN. These findings underscore the importance of raising awareness and implementing preventive measures to mitigate the risks associated with this condition.