Thalassemia is an autosomal recessive blood disorder, and this is the second most abundant genetic disorder, of which 50% of patients are from the Southeast Asian region. Beta-Haemoglobin (HBB) gene carries the mutation, and the alteration in the HBB gene sequence results in the abnormal functioning of oxygen-carrying hemoglobin molecules. This study was conducted on the 20 affected families of Thalassemia after their consent. Blood samples were collected. Then DNA was extracted using a standard organic method with some modifications. Primers for the exonic and intronic region of the HBB gene were designed and amplified following PCR protocol. Amplicons were sequenced for mutation detection using bioinformatic tools. The previous researches show that incorrect intronic and exonic splicing lead the thalassemia to reach at worst condition due to defect in mutation. The Human Splicing Finder (HSF) tool, BLAST tool from NCBI website, and Bioedit software have been used for analyzing the sequence and variations at positions. Five different samples of the HBB gene showed differences in different locations of exon and intron. HBB Exon 1 Sample 1 has intronic change with variation in base change position c269+5 on an amplified fragment from G>C. HBB Exon 1 Sample 2 has exonic change with variation in base change position c185 on an amplified fragment from T>C of nucleotide change, and codon replacement occurs at position CAT>CAC as amino acid p.His does not change. HBB Exon 1 Sample 3 has intronic change with variation in base change position c269+5 on an amplified fragment from G>C. HBB Exon 1 Sample 4 has exonic change with variation in base change position c185 on an amplified fragment from T>C of nucleotide change, and codon replacement occurs at position CAT>CAC as amino acid p.His does not change. HBB Exon 2 Sample 5 has intronic change with variation in base change position c491+16 on an amplified fragment from G>C. Graphical Representation and chromatogram of Potential splice site are showing intronic and exonic variations at the position. G replaces C in samples of intronic variation & T replaces C in samples of exonic variation. Change in sequence alignment is observed at different positions in samples of exons and introns. This study concludes that the change in the HBB gene is observed as well as intronic, and exonic splicing can lead thalassemia from severe to mild.