Sixty DNA trinucleotide cation radicals covering a large part of the genetic code alphabet were generated by electron transfer in the gas phase, and their chemistry was studied by collision-induced dissociation tandem mass spectrometry and theoretical calculations. The major dissociations involved loss of nucleobase molecules and radicals, backbone cleavage, and cross-ring fragmentations that depended on the nature and position of the nucleobases. Mass identity in dissociations of symmetrical trinucleotide cation radicals of the (XXX+2H)+• and (XYX+2H)+• type was resolved by specific 15N labeling. The specific features of trinucleotide cation radical dissociations involved the dominant formation of d 2 + ions, hydrogen atom migrations accompanying the formation of ( w 2 +H)+•, ( w 2 +2H)+, and ( d 2 +2H)+ sequence ions, and cross-ring cleavages in the 3′- and 5′-deoxyribose moieties that depended on the nucleobase type and its position in the ion. Born–Oppenheimer molecular dynamics (BOMD) and density functional theory calculations were used to obtain structures and energies of several cation-radical protomers and conformers for (AAA+2H)+•, (CCC+2H)+•, (GGG+2H)+•, (ACA+2H)+•, and (CAA+2H)+• that were representative of the different types of backbone dissociations. The ion electronic structure, protonation and radical sites, and hydrogen bonding were used to propose reaction mechanisms for the dissociations.