In most references, the transcription initiation site for the α2- and α1-globin genes has been described to lie 37 bp upstream of the translation initiation codon, however, a review of data repositories such as GenBank and Ensembl showed a report of the α2-globin transcription initiation site occurring at position -66 relative to the initiation codon. To confirm the occurrence of these isoforms for both the α2- and α1-globin genes and to document their expression levels, we initiated our current investigation. Total RNA from the peripheral blood of 15 healthy volunteers was analyzed using both semi-quantitative-polymerase chain reaction (PCR) and real-time (ReTi-PCR) protocols developed in our laboratory, with primers designed to enable distinction between the α2- and α1-globin transcripts.We observed two distinct PCR products for each of the globin genes. Subsequent DNA sequencing of 11 individual PCR products revealed that the α2- and α1-globin transcripts are present in both a long and a short isoform, initiating at positions -66 and -37, respectively. The shorter (-37) isoform is expressed approximately 10,000-100,000 times more strongly than the longer isoform, demonstrating differential expression within the healthy population. This study, for the first time, confirms the presence of two isoforms for both the α2- and α1-globin genes with varying transcription levels in healthy individuals. The short isoform is expressed at significantly higher levels than the longer isoform for both α2 and α1 genes. Therefore, based on our observations, we propose that despite the contribution of the long isoforms to the total α-globin RNA pool, the short isoforms are the main physiological transcripts.