Background: Thalassemia is a prevalent condition characterized by decreased production or absence of hemoglobin globin chains. There are 2 types of thalassemia, alpha thalassemia and beta thalassemia (β-thalassemia), classified according to defects in the alpha or beta protein chain. β-Thalassemia results from a diverse range of mutations in the β subunit of the hemoglobin gene (HBB). This results in severe reductions in erythrocyte and hemoglobin levels. Worldwide, more than 40,000 babies are born with β-thalassemia annually. There are approximately 80 million β-thalassemia carriers. In Pakistan, the annual birth rate of β-thalassemia major carriers is approximately 6,000, and 50,000 to 100,000 patients suffer from this common inherited disorder. IVSI-1, IVSI-5 (G>C), Fr 8/9 (+G), Fr 41/42 (-TTCT), and del 619 are the 5 most common mutations that account for 90% of β-thalassemia cases in Pakistan. This study aimed to investigate the prevalence of β-thalassemia mutations (IVSI-5, Fr 41/42, and Fr 8/9) in the Swat district and to analyze the blood morphology of different mutant thalassemia blood samples. Methods: A cross-sectional study was performed at the Forensic Research Lab University of Swat from December 2021 to March 2022. The study included a total of 150 blood samples. Comparative analysis of blood morphology through microscopy was carried out on samples from patients with different β-thalassemia mutations and normal individuals. Genomic DNA extraction was performed according to the Sambrook protocol, and DNA was quantified via agarose gel electrophoresis. Thalassemia mutations were detected by amplification refractory mutation system (ARMS)-polymerase chain reaction (PCR). The frequencies of different β-thalassemia mutations were analyzed. Results: Thalassemia blood cells were more hypochromic and microcytic than normal blood cells, although no differences were detected in the morphology of IVSI-5, Fr 8/9, or CD41/42 mutant blood cells. The IVSI-5, Fr 8/9, and CD41/42 mutations were analyzed through agarose gel electrophoresis of the ARMS-PCR data. The IVSI-5 mutation was the most prevalent mutation, identified in 52.2% (78/150) of the patients. The prevalence of Fr 8/9 and CD41/42 mutations was 22.6% (34/150) and 18.6% (28/150), respectively. A total of 5.33% (8/150) of patients had double mutations, and only 1 patient (0.666%) had all 3 mutations. Conclusion: This study provides a reliable reference for the detection of different mutations in β-thalassemia and will help to formulate different strategies for improving the awareness of the general public. For a more thorough analysis of common β-thalassemia mutations, the investigation should be extended to more ethnic regions of Pakistan.