The molecular basis of β-thalassaemia in two linguistically and ethnically different groups of Pakistan was studied. These groups were the Potwari Punjabis residing in the Potwar region of North West Punjab and the Pathans residing in the North West Frontier Province (N.W.F.P.). A combination of amplification-refractory mutation system-polymerase chain reaction (ARMS-PCR) and DNA sequencing was used to screen for mutations in the β-globin gene of patients and carriers. In the Punjabis three mutations; IVS-1 position 5 (G-C), codons 8/9 (+G) and codons 41/42 (-CTTT) account for 82.2% of the total characterized alleles. In this groups the rare mutations, found at 17.8% of the total characterized alleles, are capsite +1 (A-C), codon 16 (-C), codon 5 (-CT), the 619 bp deletion at the 3′ end of the gene, IVS-2 position 1 (G-A) and codon 30 (G-C). In the Pathans four mutations; codons 8/9 (+G), IVS-1 position 5 (G-C), codon 5 (-CT) and codons 41/42 (-CTTT) account for 82.4% of the total characterized alleles. the rare mutations in this group account for 17.5% of the total characterized alleles and are codon 15 (G-A), codon 16 (-C), capsite +1 (A-C), IVS-1 position 1 (G-T), codon 30 (G-A), IVS-1 position 1 (G-A), codon 30 (G-C) and codon 6 (A-T). the mutation spectrum reproted in this study differs from that reported previously by Ahmedet al.(1997, British Journal of Haematology,94, 476–482) in Punjabis and Pathans. the differences in the mutation spectrum of the β-globin gene in thalassaemia patients and carriers in the Potwari Punjabis and the Pathans reported in the present study emphasizes the importance of determining and classifying accurately the ethnic history of the subjects and the mutations present in each ethnic group within a country separately.