Impatiens L. is a genus of complex taxonomy that belongs to the family Balsaminaceae (Ericales) and contains approximately 1000 species. In this study, ISSR, trnL-trnnF, atpB-rbcL and DEF four molecular markers were used to analyze the genetic diversity and phylogenetic relationships among 32 species of Impatiens from eastern Yunnan. The results showed that the genetic similarity coefficient (GS) of these Impatiens resources ranged from 0.540 to 0.990, it indicated that the genetic diversity was relatively rich. However the genetic distance was relatively vague, and the evolutionary relationship was not obvious, which the phylogenetic trees were constructed by using the chloroplast genes atpB-rbcL and trnL-trnF. The genetic distance of the phylogenetic tree constructed by the DEF gene was relatively clear, the evolutionary relationship was obvious, and the result was consistent with ISSR. This indicated that DEF gene was greatly affected by plant habitat, and had abundant variation and a fast evolution rate, while atpB-rbcL and trnL-trnF genes had a slower evolution rate. And it was discovered that the flower morphological traits of the genus Impatiens can be employed as significant categorization markers. In particular, the wing shape can be used as an important flower morphological feature for the classification of the genus Impatiens in eastern Yunnan; and the results of using ISSR and DEF gene markers and flower morphological characteristics were consistent with the results of the interspecies division of Impatiens in this region. This study provided new basic data and a scientific basis for the protection, sustainable development, and utilization of Impatiens plant resources and genetic breeding in this region.