Aconite alkaloids from the roots of Aconitum carmichaeli (Fuzi, in Chinese) have been investigated by rapid-resolution liquid chromatography coupled with time-of-flight mass spectrometry (TOFMS) in positive mode. With dynamic adjustment of the key role as fragmentor voltage in TOFMS, an efficient transmission of the ions was achieved to obtain the best sensitivity for providing the molecular formula for each analyte, and abundant fragment ions for structural information. Fifteen authentic standards isolated from Fuzi with various structures were first characterized by TOFMS, including diester-diterpenoid alkaloids (DDAs), monoester-diterpenoid alkaloids (MDAs), alkylol amine-diterpenoid alkaloids (ADAs), veatchine-type alkaloids and atisine-type alkaloids. Fragmentation rules and key diagnostic fragment ions have been summarized, and possible pathways of fragmentation have been proposed. By accurate mass measurements within 5 ppm error for each ion, 30 C(19)-diterpenoid alkaloids including 10 DDAs, 3 MDAs, 9 ADAs and 8 other type alkaloids, and 8 C(20)-diterpenoid alkaloids including 4 veatchine-type alkaloids and 4 atisine-type alkaloids could be identified in a methanolic extract of Fuzi. Some isomers of aconite alkaloids were also differentiated. Based on the differences between their fragmentation pathways and special fragment ions, each type of aconite alkaloids was differentiated.