Background: Hemoglobin A2' (delta 16 Gly → Arg) is globally the commonest delta chain variant of HbA2. It is clinically and hematologically silent but its sole importance lies in the underestimation of HbA2 quantity during the workup of ββ-thalassaemia trait. High performance liquid chromatography (HPLC) identifies it as a small S-window peak with a mean retention time of 4.59 ± 0.03 minutes. This study aims at describing the frequency of detection of HbA2' by HPLC in Pakistan and its confirmation at a molecular level. Potential HbA2' cases were identified by a retrospective review of 10186 HPLC chromatograms in year 2006. Prospective samples were collected for polymerase chain reaction (PCR) amplification, restriction digestion and nucleotide sequencing. Findings: One hundred and ninety two potential cases (1.89%) of HbA2' were detected on HPLC, having mean retention time of 4.59 ± 0.05 minutes. Sixty four (0.6%) new cases were suspected of having co-existing ββ-thalassaemia trait when the quantity of S-window peaks was taken into account. Thirteen samples with presumed HbA2' on HPLC were subjected to molecular analysis and the said mutation (δ 16 GGC → CGC) was not detected in any sample. Conclusions: It is concluded that diagnosis of HbA2' on HPLC alone is not justified, as evidence of the presence of this delta chain variant in Pakistani population is yet to be proven. Such small S-window peaks should be either disregarded or confirmed at molecular level, and only then should influence the diagnosis of β-thalassaemia trait. Further studies are required to determine the true nature of these peaks.