Analysis of mitragynine in different species of mitragyna, indigenous plant of peninsular Malaysia is an important area of research due to the use of this compound as a drug of abuse. A fast, selective and reproducible ultrasonic assisted extraction (UAE) coupled with gas chromatography-mass spectrometry (GC-MS) method was developed for the determination of mitragynine in different species of mitragyna (mitragynaspeciosa and mitragynarotundifolia). Three samples of mitragyna species, namely KM, KY1 and KY2 were collected from Kedah state of Malaysia and identified as a mitragynine-containing species which are authenticated by botanists. UAE was carried out with chloroform-methanol (1:4-v/v) as extracting solvent at 30°C and 1.0 h sonication time. The GC-MS analyses of extracted samples confirmed the presence of mitragynine in KM and KY1 samples, while mitragynine was not detected in KY2 samples. Under optimized conditions, the quantitative estimation of mitragynine in KM and KY1 samples were found to be 0.094% and 0.105%, respectively.