Methylamphetamines (methylamphetamine (Me-AP) and methylmethamphetamine (Me-MA)) have three ringpositional isomers. Among them, 4-Me-AP has been controlled as a designated substance (Shitei-Yakubutsu) in Japan since November 2012. Furthermore, Me-APs are a structural isomer of methamphetamine (MA), controlled as a stimulant. Because of the increasing number of structural isomer of designer drugs encountered in forensic science laboratories, analytical differentiation of structural isomers is a significant issue. In this study, a method for differentiation of methylamphetamines using gas chromatography/mass spectrometry (GC/MS) was developed. DB-1ms and DB-5ms columns could not separate free bases and trifluoroacetyl (TFA) derivatives of methylamphetamines while the columns incompletely separated their trimethylsilyl (TMS) derivatives. On the other hand, the mid-polar DB-17ms column separated free bases, TFA and TMS derivatives of 6 methylamphetamines though peak shapes of the free bases were tailing. The best separation was obtained from the analysis of the TMS derivatives on DB-17ms column. The mass spectra showed a little difference between the 2-, 3-Me-AP and 4-Me-AP after TFA derivatization. Also the structural isomers of Me-AP and MA could be differentiated by their EI mass spectra. The results indicated that differentiation of regioisomeric methylamphetamines could be accomplished well by GC/MS of their TMS derivatives on the mid-polar capillary column.