Telomeric DNA has been intensely investigated for its role in chromosome protection, aging, cell death, and disease. In humans the telomeric tandem repeat (TTAGGG)n is found at the ends of chromosomes and provides a novel target for the development of new drugs in the treatment of age related diseases such as cancer. These telomeric sequences show slight sequence variations from species to species; however, each contains repeats of 3 to 4 guanines allowing the G-rich strands to fold into compact and stable nuclease resistant conformations referred to as G-quadruplexes. The focus of this manuscript is to examine the effects of 5′-nucleotides flanking the human telomeric core sequence 5′-AGGG(TTAGGG) 3-3′ (h-Tel22). Our studies reveal that the addition of the 5′-flanking nucleotides (5′-T, and 5′-TT) results in significant changes to the thermodynamic stability of the G-quadruplex structure. Our data indicate that the observed changes in stability are associated with changes in the number of bound waters resulting from the addition of 5′-flanking nucleotides to the h-Tel22 sequence as well as possible intermolecular interactions of the 5′ overhang with the core structure. © 2014 by the authors; licensee MDPI, Basel, Switzerland.