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Internal transcribed spacer (ITS) regions and maturase K (matK) sequence polymorphisms provide an efficient tool for discrimination and conservation of genetic resources of Anoectochilus species. The objectives of this study were to develop markers specifically distinguishing A. formosanus Hayata from closely related A. koshunensis Hayata, A. roxburghii (Wall.) Lindl., and Ludisia discolor (Ker Gawl.) A. Rich. and to identify a molecular phylogenetic relationship of a new intergeneric BC1F1 hybrid - Ludochilus Jin-Chai. Specific primers for nuclear ITS regions and chloroplast matK sequences were designed and converted into cleaved amplified polymorphic sequence (CAPS) markers. Results show that the matK sequences obtained corresponded to pseudogenes and that their digestion with enzyme HinfI revealed a polymorphic pattern in A. formosanus and A. koshunensis. The pedigree of Lud. Jin-Chai, which was derived from the cross between Lus. discolor and A. formosanus, was also confirmed based on ITS and matK CAPS markers.