Quantitative structure-retention relationships studies were performed for cholesterol and alkylamide stationary phases, which were previously applied in the analysis of nucleotides and oligonucleotides. An octadecyl column was also tested. Twenty-four oligonucleotides of various sequences and length were chosen; next, their structural descriptors were determined with the use of quantum-mechanics method. The sequence features were related mainly to their surface area, hydrophobicity, and the nature of nucleobases. Moreover, for the first time models employing experimentally derived descriptors (the sum of retention factor for individual nucleotides) were developed in the quantitative structure-retention relationship studies of these compounds. The retention of oligonucleotides for alkylamide and cholesterol stationary phases may be effectively predicted with the use of quantitative structure-retention relationship models based only on molecularly modeled descriptors, as well as with models employing experimentally derived descriptors. Therefore, we recommend the first approach, since descriptors may be easily and quickly calculated. However, oligonucleotide retention prediction for octadecyl phases gives better results, when individual nucleotide retention factors are known and utilized for the creation of a mathematical model.