Unexpectedly, the 5‐dehydroquinoline radical cation was formed in the gas phase from the 5‐iodo‐8‐nitroquinolinium cation upon ion‐trap collision‐activated dissociation. This reaction involves the cleavage of a nitro group to generate an intermediate monoradical, namely, the 8‐dehydro‐5‐iodoquinolinium cation, followed by rearrangement through abstraction of a hydrogen atom from the protonated nitrogen atom by the radical site. Dissociation of the rearranged radical cation through elimination of an iodine atom generates the 5‐dehydroquinoline radical cation. The mechanism was probed by studying isomeric biradicals and performing quantum chemical calculations. The 5‐dehydroquinoline radical cation showed greater gas‐phase reactivity toward dimethyl disulfide, cyclohexane, and allyl iodide than the isomeric 5,8‐didehydroquinolinium cation, which is more reactive than the isomeric 5,8‐didehydroisoquinolinium cation studied previously. All three isomers have a 1,4‐biradical topology. The order of reactivity is rationalized by the vertical electron affinities of the radical sites of these biradicals instead of their widely differing singlet–triplet splittings.