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Ecotypes are the result of ecological differentiation at the early stages of speciation. Adaptation to soil conditions offers arguably the best examples of local adaptation in plants. Two sympatric ecotypes, with either a red or green abaxial leaf surface, were found without clear geographical isolation in Phalaenopsis pulcherrima, a Southeast Asia endemic and endangered orchid. The soil of the red leaf ecotype has a higher water content and nutrient content than the green ecotype. What is the genetic structure of the two ecotypes? Is there complete or partial reproductive isolation between the two ecotypes? In this work, leaf reflection of the two ecotypes in P. pulcherrima were compared, to illustrate their difference in leaf color. The genetic differentiation between two ecotypes was examined, using ISSR and SRAP markers to determine the genetic structure of the populations. Our results showed that the green ecotype had reflectance spectrum peaks at 530 nm and 620 nm, while in the red ecotype, the peak at 530 nm was absent. A total of 165 ISSR and SRAP loci showed a high level of genetic diversity within the green ecotype, and analyses of the population structure revealed two genetic clusters that corresponded to the red and green ecotypes. The percentage of variation between the two ecotypes (24.55%) was greater than the percentage of variation among the populations (16.54%)—indicating partial reproductive isolation, high genetic differentiation, and that ecological differentiation has been more important than geographical barriers among populations within ecotypes. Most pairwise FST values between the populations within either ecotype on Hainan Island were less than 0.15; however, the FST between both the Thai and Malaysian populations and the Hainan Island population was greater than 0.25, due to South China sea isolation. Ecotypic differentiation is an important part of speciation; therefore, we must take into account the axes along which lineages sort, when formulating protection strategies. [ABSTRACT FROM AUTHOR]