Studies were undertaken to explore the application of tandem mass spectrometry for the structure analysis of unfractionated mixtures of oligonucleotides. Limited combinatorial libraries were constructed of mixtures of 8-mers (NGACACNG; nine compounds) and 12-mers (NGACTNAGACNG; 27 compounds), where N is any of the 2’-deoxyribonucleotides of uracil, thymine, or 5-[N-(aminoethyl)-3-acrylimido]uracil. Molecular mass measurements of the mixture components (single mass analyzer) in the simplest cases or acquisition of collision-induced dissociation mass spectra (tandem mass analyzers) for mixtures of sequence isomers of the same molecular mass were used to establish guidelines for structure assignments. Although the construction of mass sequencing ladders from gas phase backbone cleavage reactions is notably more complex in the case of isomeric mixtures than for the single isomer case, assignment ambiguities are reduced by the presence of nonrandomized sequence positions and by recognition of likely fragment ion relative abundances. The approaches described offer a significant advance toward solving problems common to the development of oligonucleotide therapeutics using combinatorial synthesis techniques.