Sickle cell anemia (SCA) is one of the most common hematologic diseases affecting humans. Detection of a single base pair mutation at 6th codon of β-globin gene is important for the diagnosis of SCA. The aim was to study the nucleotide sequences and the molecular survey of β-globin gene in Saudi patients.Blood samples from 77 unrelated SC patients were obtained from the KKUH, between 2015 and 2017. In this study, DNA was extracted then PCR was performed. Twelve overlapping fragments covering β-globin gene, have been generated by PCR.A total of 47 alterations have been recognized in β-globin gene. These alterations composed of: deletions, insertion or substitutions as follows:- one mutation identified on the 1st segment; three alterations on 2nd fragment; two alterations on 3rd segment; seven alterations on 4th segment; three substitution on 5th fragment; two changes on 6th fragment; five alterations on 7th fragment; seven substitution changes on 8th fragment; two heterozygous substitution changes on 9th fragment; three changes on 10th fragment and eight substitution changes on 11th fragment, and four changes on 12th fragment.SCA had profound negative effects on many organs, causing many complications. The results should be taken further to set up management strategies to improve outcomes.