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Objective The purpose of this study was to perform hematological and molecular analyses of the HbS allele of the hemoglobin subunit beta gene in the Sudanese population. Methods This was a descriptive cross-sectional study. Hematological parameters and fetal hemoglobin (HbF) levels were assessed in all participants. Data were gathered through the use of questionnaires and laboratory investigations. The βS-globin haplotypes, S allele distributions, and hematological parameters with HbF levels were investigated using PCR-restriction fragment length polymorphism, gel electrophoresis, and a Sysmex hematology analyzer, respectively. Results According to our findings, the Bantu (BA) haplotype was found in 10.8% of participants with homozygous uncontested haplotypes, followed by Benin (BA) and Sudan (SU), each in 9.8% of participants. This Sudanese group from Northern Kordofan lacked the Arab-Indian haplotype. Two heterozygous versions of undisputed haplotypes were found in 17.3% of participants: SU/BA in 10.8% and CA/BE in 6.5%. Conclusion As a result of sickle cell anemia, this investigation found changes in hematological parameters. In the Sudanese population, a new haplotype of the S gene was discovered.