β-Thalassemias comprise a group of heterogeneous hemoglobin (Hb) disorders characterized by the absence or reduced synthesis of the β-globin chain with a variable clinical presentation. The Al-Qatif and Al-Ahsa oases in the Eastern Province of Saudi Arabia are regions known for the high prevalence of these disorders. This study was conducted to provide a more precise picture of the β-thalassemia (β-thal) mutations prevalent in these regions and to estimate their frequencies. One hundred and 96 subjects with transfusion-dependent β-thalassemia (β-thal) disease were included in this study. A total of 14 β-thal mutations were identified with five mutations accounting for more than 80% of the total β-thal mutations identified. Of the 196 patients, 164 were homozygous for a β-thal mutation, while 32 were compound heterozygotes. We report here the novel identification of two mutations, namely, the Tunisian splice site IVS-I-130 (G→C) and the Mediterranean cryptic splice site IVS-I-110 (G→A), which have not been previously reported in the population of the Eastern Province. However, 15 patients (46.9%) with compound heterozygosities carried one of the β-thal mutations and the sickle cell mutation [Hb S or β6(A3)Glu→Val]. These patients were less frequently transfused than the patients who were homozygous for the β-thal mutations and presented with fewer complications. A more comprehensive overview of the genetic heterogeneity of the β-thal mutations in the Eastern Province of Saudi Arabia is presented in this article. This study will contribute to the establishment of an effective prevention program, including premarital screening.