To study the quality and genetic diversity in Gentiana macrophylla of different habitats for controlling the quality of Gentiana macrophylla.Main characteristics and microscopic identification were adapted to identify Gentiana macrophylla; HPLC method was applied for analysising the contents of Gentiana macrophylla. RAPD method was applied to study genetic structure and genetic diversity of Gentiana macrophylla.The experiment showed that the Gentiana macrophylla of different habitats have different main charactericstics, microscopic identifications and contents, and effective ingredient contents of gentiopicroside. The levels of population genetic diversity of various groups followed by HUANGZHONG County population (K) > HUAN County population (J) > ZiWU Mountain population (H).The study of external morphology, internal structure, active ingredient content and genetic level of Gentiana macrophylla from different areas provides a scientific basis for protecting wild species and ensuring the quality in introducing and cultivating Gentiana macrophylla.