DNA and locked nucleic acid (LNA) were characterized as single strands, as well as double stranded DNA‐DNA duplexes and DNA‐LNA hybrids using tandem mass spectrometry with collision‐induced dissociation. Additionally, ion mobility spectrometry was carried out on selected species. Oligonucleotide duplexes of different sequences – bearing mismatch positions and abasic sites of complementary DNA 15‐mers – were investigated to unravel general trends in their stability in the gas phase. Single stranded LNA oligonucleotides were also investigated with respect to their gas phase behavior and fragmentation upon collision‐induced dissociation. In contrast to the collision‐induced dissociation of DNA, almost no base loss was observed for LNAs. Here, backbone cleavages were the dominant dissociation pathways. This finding was further underlined by the need for higher activation energies. Base losses from the LNA strand were also absent in fragmentation experiments of the investigated DNA‐LNA hybrid duplexes. While DNA‐DNA duplexes dissociated easily into single stranded fragments, the high stability of DNA‐LNA hybrids resulted in predominant fragmentation of the DNA part rather than the LNA, while base losses were only observed from the DNA single strand of the hybrid.