Objective To study the genetic diversity and genetic relationship among different habitats and growth patterns of Anoectochilus roxburghii. Methods A total of 20 fresh leaves of Fujian Anoectochilus roxburghii were used as samples, and 15 ISSR primers with good polymorphism and amplification were screened out by ISSR-PCR molecular marker technology. After the agarose gel electrophoresis, the number of amplified bands was counted. The data were analyzed by NTSYS-Pc 2.1 and POPGENE 32 software, and clustering were performed by UPGMA method. Results A total of 130 clearly visible bands were amplified, and the polymorphism ratio is up to 100%. The genetic similarity coefficient ranged from 0.647 6 to 0.971 4, and the genetic distance distribution was 0.029 0 to 0.434 5. The genetic relationship between samples No.3 and No.4 was the closest. The clustering results showed that when the similarity coefficient is 0.78, 20 samples were divided into three categories, which could distinguish between different habitats and varieties. Conclusion The ISSR marker technology could reveal the genetic diversity of Anoectochilus roxburghii at the molecular level.