The Direct Amplification of Length Polymorphisms was applied to assess genetic diversity and structure of 7 populations of Dendrobium nobile, comparing one population of D. lituflorum. The five primer combinations were amplified to produce 140 clear bands, and 102 polymorphic bands had been detected with each pair of primer producing 20.4 polymorphic bands on average. At species level, the percentage of polymorphic bands (PPB) was 72.86%, the Nei's gene diversity index (H) was 0.288 9, and the Shannon's information index (I) was 0.424 2. At population level, the average PPB was 47.96%, H was 0.1861, and I was 0.273 9 in 7 populations. The coefficient of gene differentiation (Gst) was 0.338 6 among populations of D. nobile. It showed that 33.86% of the total genetic diversity was attributable to genetic differentiation among populations, while the rest 66.14% was resided between individuals within population.