This study aimed at the identification of the spectrum of mutations in patients with β-thalassemia (β-thal) in the western province of Saudi Arabia. Screening for the mutations was done using the polymerase chain reaction-amplification refractory mutation system (PCR-ARMS) technique to test for 12 mutations, and direct automated DNA sequencing for the unknown samples. The study included 172 patients; of these 15 patients had sickle cell anemia and one Hb S [β6(A3)Glu→Val, GAG>GTG]/β-thal. A total of 23 mutations were identified to cause the disease in the western area. Seven common mutations were responsible for the β-thal alleles in 78% of patients and could be detected by the ARMS technique: IVS-II-1 (G>A), IVS-I-110 (G>A), IVS-I-5 (G>C), codon 39 (C>T), codon 26 (G>A) [Hb E or β26(B8)Glu→Lys, GAG>AAG], frameshift codons (FSC) 8/9 (+G), and IVS-I-1 (G>A). DNA sequencing of uncharacterized alleles detected eight less common mutations: FSC 41/42 (-TCTT), IVS-I 25 bp deletion, codon 37 (G>A), FSC 44 (-C), Cap site +1 (A>C), IVS-I-6 (T>C), FSC 5 (-CT) and IVS-I-1 (G>T), and eight rare mutations: -87 (C>G), initiation codon -1 (T>G), codon 15 (G>A), FSC 16 (-C), FSC 20/21 (+G), codon 27 (G>A), IVS-I-130 (G>C) and IVS-II-837 (A>C). Four alleles were normal by DNA sequencing. Genetic heterogeneity was observed in this study, 10 mutations were of Asian or Asian/Indian origin, two were Kurdish, one Chinese, one Turkish, one Saudi, and the remainder were of Mediterranean origin. The presence of a large population of immigrants in the western province is responsible for the great heterogeneity at the molecular level, and for the difference observed in the frequencies of mutations from those reported in the eastern province of Saudi Arabia. Screening for β-thal mutations using PCR-ARMS for the seven most frequent mutations in the Saudi population followed by DNA sequencing of the unknown alleles could be useful for the implementation of a strategy for carrier detection and preimplantation genetic diagnosis in high risk families.