The phenotype of increased Hb A2 typical of β-thalassaemia (β-thal) carriers can be reduced to normal or borderline values because of the co-inheritance of a δ-globin gene (HBD, MIM #142000) mutation, which may lead to misinterpretation of diagnostic results. To know the spectrum of δ-globin mutations in the Portuguese population we performed a mutational analysis of the δ-globin gene in a group of 51 Portuguese β-thal carriers presenting microcytosis, hypochromia and a normal/borderline Hb A2 level and in another group of 15 individuals suspected to have δ-globin structural abnormalities. The heterozygosity for the βIVS-I-6T→C (HBB:c. 92+6T>C) mutation was the main cause for the mentioned atypical β-thal carrier phenotype. Furthermore, eight individuals were double heterozygous for one common β-thal mutation and the δCd27G→T mutation (Hb A2–Yialousa; HBD:c.82G>T). One of them also presented a novel δ-globin gene promoter mutation,−80G→A (HBD:c.−130G>A), responsible for about 25% decrease of the promoter activity in transient expression assays. One the other hand, in the other group of 15 individuals suspected to have δ-globin structural abnormalities observed by biochemical methods, some known Hb A2 variants were identified – Hb A2′ (HBD:c.49G>C), Hb A2-Babinga (HBD:c.410G>A), and Hb A2-Wrens (HBD:c.295G>A), and the novel Hb A2-Fogo [δ64(E8)(Gly→Ser); (HBD:c.193G>A)]. This novel Hb A2 variant was observed segregating in linkage with Hb E (HBB:c.79G>A) in a three generation family. In conclusion, six different δ-globin mutations were found, being two of them new molecular defects. All δ-alleles identified were found linked to the expected β-globin cluster haplotype. All mutations caused a low Hb A2 level and through this could lead to misdiagnosis when inherited together with a β-thal allele.