In the 5' untranslated region (5'UTR), which is transcribed but not translated and is involved in posttranscriptional regulation of the gene promoting and stabilizing the mRNA translation, several mutations associated with mild β-thalassemia (β-thal) have been described. One of these, the +20 (C>T) mutation, is described in the HbVar database as a mutation responsible for β(+)-thal. In heterozygote cases, it gives rise to a phenotype of β-thal minor and β-thal intermediate (β-TI) when the mutation is associated with β(+) IVS-II-745 (C>G). To clarify if this mutation is responsible for β(+)-thal, we studied nine cases where we found an association of the +20 and IVS-II-745 mutations. All patients were carriers of four α genes. Three patients carried β-thal major (β-TM), two were compound heterozygotes for IVS-II-745 and codon 8 (-AA) or codon 39 (C>T), and the third was homozygous for IVS-II-745; all had the +20 mutation in the 5'UTR. The remaining patients showed the mutation IVS-II-745 associated with a replacement of C>T at nucleotide (nt) +20 of the 5'UTR. Contrary to reports in the HbVar database, the +20 mutation should be considered as an innocuous single nt polymorphism associated with the IVS-II-745 mutation in cis.