Abstract: This work describes the basic physicochemical properties of phosphoryl guanidine oligonucleotides (PGOs)—a new type of DNA analog with a partially or totally uncharged backbone. Replacement of the negatively charged oxygen atom with an electroneutral 1,3-dimethylimidazolidine-2-imine (DMI) group is shown to influence the hydrophobicity of oligonucleotide, its electrophoretic mobility, and UV absorption spectra. Thermal stability of DMI-containing DNA-duplexes was studied in aqueous solutions with different ionic strength. It is determined that, in the case of low ionic strength, the presence of DMI groups significantly enhances the thermal stability of the duplex. In the case of nearly physiological condition (0.1 M NaCl, 10 mM MgCl2) or higher ionic strength, DMI groups do not affect the duplex stability or even slightly reduce it (melting temperature changes, on average, by –1.2°C per one DMI group). An important feature of totally uncharged PGO is its ability to form the duplex effectively regardless the ionic strength, even in deionized water. In addition, according to CD data, DMI groups do not disturb the double helix structure of DNA, maintaining it in the B form.