Alpha-thalassemia is an inherited blood disorder which is an autosomal recessive type disorder characterized by a microcytic hypochromic anemia and hemolytic anemia. The a-thalassemias involve the genes?HBA1?and?HBA2.The aim of this research was to determine the mutations in hemoglobin alpha 1 (HBA1) in Thalassemia affected patients and in silico analysis of identified mutations to predict the functional effect. In this study, genomic DNA was extracted from 40 Patients affected with Thalassemia (n=40) disease. Blood samples were collected in vacutainers with EDTA as an anticoagulant from the patients and relatives. Blood samples with anticoagulant were used for leukocytes based DNA extraction. Standard organic method was used for DNA extraction. DNA samples were quantified using agarose gel and DNA ladder. Primers were designed using gene sequence from NCBI gene bank. Primer3 software was used for primer designing. PCR conditions will be optimized for amplification and PCR was performed to determine the SNPs. A 382 base pair fragment of DNA of HBA1gene of exon 3 was amplified using polymerize chain reaction (PCR) technique. Sanger sequencing of the selected samples was done to identify polymorphisms. A total of 24 samples out of 40 samples of DNA were sequenced and these SNPs were confirmed by alignment. We were unable to find the mutations in the HBA1 gene but two heterozygous variations were found in HBA1exon 3.Two heterozygous variations were confirmed in exonic area of HBA1 gene of Patients affected with Thalassemia. The findings of this research revealed no mutations were found in HBA1 gene. Two heterozygous variants were confirmed at the position of c.514 on amplified fragment from G> C and second change at the position of c.470 on amplified fragment G > C in 3?UTR.. Variations were further subjected for splice site analysis. The splicing site analysis was done by using an online tool (Human splicing finder).A variation which were present at c.514 G>C were found in the potential splice site and its consensus value is 88.39 and the second one is not in the target region of splicing.