Abstract: The relative content and structure of components of the essential oil from Mentha rotundifolia (L.) Huds. growing in Guizhou was analyzed and identified using headspace solid phase-microextraction gas chromatography-mass spectrometry (HS-SPME-GC-MS). As a result a total of 38 chemical components were identified from the volatile oil of Mentha rotundifolia (L.) Huds. in Guizhou. The main chemical components are D-carvone (relative content 51.78%) and D-limonene (relative content 20.93%). The HS-SPME-GC-MS shows the good characteristics of fast, comprehensive and accurate in the analysis of volatile oil of the Mentha rotundifolia (L.) Huds., indicating that it is very suitable for the analysis and identification of the essential oil of this species.