Vertical transmission remains the leading cause of Human Immunodeficiency Virus (HIV) infection among children under 15 years worldwide. This transmission occurs during pregnancy, labor, delivery, or breastfeeding. Preventing this form of transmission is crucial in reducing pediatric HIV cases. Objective: This study aimed to assess the effectiveness of an educational program on midwives' knowledge regarding the prevention of vertical transmission of HIV. Methods: The study used a cluster randomized design enrolling two clusters of health centers(( the south and west clusters of Health Centers and internally displaced person camp clinics closed to Alfasher Town, to access the education information to a large number of study samples) the targeted populations included all midwives (169) working at two clusters of health centers and. Internally displaced persons camp Clinics), simple random sampling was used to allocate participants to intervention and control arms. The study targeted 169 midwives but 159 participants were participated they recruited from the 12 Health Centers and 9 internally displaced persons camp clinics),included (6 Health Centers +5 internally displaced persons camp clinics intervention arm) and (6 Health Centers +4 internally displaced persons camp clinics control arm). The data were collected using a self-administered questionnaire; the completed questionnaires were carefully checked for completeness to ensure high-quality data. The data were analyzed using the Statistical Package for the Social Sciences (SPSS) version 20, with 95% Confidence Intervals (CIs), and P-values < 0.05 were considered as significant The results were summarized in frequency, percentage, and mean scores and presented in tables. The training intervention was simple, and exposed the intervention arm to education sessions about; HIV/AIDS, vertical transmission, and strategies for prevention of vertical transmission, the session was conducted at a midwifery school over 2weeks; 3/lectures/week, the session 3hours\day inform of lectures through multimedia and role-play, group discussion, the sessions conducted by researcher and 2 assistant (reproductive health (RH) coordinator and head of midwifery office in State Ministry of Health). Although the control group did not received training intervention. The ethical approval was obtained from the Institutional Review Board at Al Neelain University and the Ministry of Health, North Darfur State. Written consent was obtained from each participating midwife. Results: The majority of midwives are between 25 and 30 years old. Educational qualifications showed that most participants were midwives, with only 5 (6.3%) holding a Bachelor of midwifery degree. The educational program significantly improved midwives' knowledge regarding HIV vertical transmission prevention, and that intervention group showed significant improvements across all dimensions of knowledge. The mean knowledge score about vertical transmission in the intervention group was 8.18 (SD = 1.6), compared to 3.1 (SD = 1.6) in the control group, with a P-value of 0.00. Similarly, knowledge about the prevention of vertical transmission was 8.23 (SD = 1.6) in the intervention group, versus 3.1 (SD = 1.5) in the control group, with a P-value of 0.00. The overall mean knowledge score among the intervention group was 25.24 (SD = 3.3), while the control group had a mean score of 12.1 (SD = 3.1), with a P-value of 0.00. Conclusion: The findings highlight that the intervention program had a positive impact on the participants' knowledge that showed a significant improvement among intervention group compared to the control group; they demonstrated inadequate knowledge towards MTCT of HIV/ AIDS so, the study recommends that comprehensive training of midwives in vertical HIV prevention was a beneficial program and positively impacted their knowledge. Key words: midwives, knowledge, vertical transmission, prevention, HIV, Internally displaced persons. Educational program, Sudan