Using Anoectochilus roxburghii plants with or without leaf veins from ‘Wuyi No. 1’ germplasm resource of Wuyi Mountain, Fujian Province as materials, we carried out trait observations and medicinal component analysis in this study. Artificial crossing was also carried out with these two kinds of plants as the parent. The pattern of hybrid progeny segregation was analyzed and the parents and hybrid progeny were identified by ISSR and CDDP molecular markers. Our studies provide the basis for cultivar improvement of the Fujian native A. roxburghii germplasm resources. The results showed that: (1) flavonoid and total polysaccharide contents in the plant with leaf veins were 1.2 times and 1.3 times as high as in the plant without leaf veins, respectively. However, the content of kinsenoside in the plant without leaf veins was slightly higher than the plant with leaf veins. (2) Hybridization results showed that the hybrid compatibility of the parent with the same leaf vein trait was higher than the parent with different leaf vein trait. The segregation ratio of with and without leaf veins hybrid progeny was about 3∶1. We infer that the genotype with leaf veins is homozygous and the genotype without leaf veins is heterozygous. (3) Plants with or without leaf veins can be effectively distinguished by combined application of ISSR and CDDP molecular markers, ISSR and CDDP markers can also be used to identify hybrid progeny. Cluster analysis showed the genetic distance of plants with and without leaf veins was 0.04. Our results indicate that there are significant differences in the contents of medicinal components among different strains of Fujian native A. roxburghii germplasm resource and support the view that A. roxburghii with and without leaf veins are different strains.