Non-coding regions of the chloroplast genome offer interesting levels of nucleotide variation which are very useful for molecular genetics, population and phylogenetic analysis. The family Orchidaceae is represented by ca. 500 species in Southern Ecuador. In order to determine the genetic variability present in members of this family belonging to the genera Cyrtochilum, Masdevallia, Epidendrum, Polystachya, Stelis and Zelenchoa , we have analyzed four chloroplastic intergenic spacers: atpH - atpI, trnL - trnF, trnF- ndhJ and rps16 - trnQ. All these markers have shown high richness in simple sequence repeats (SSR), indels and substitutions. They resulted to be useful for species identification, phylogenetic analysis and population structure studies. Moreover the information provided by this analysis suggests that the endemic species Masdevallia deformis must be considered vulnerable and conservation strategies need to be adopted for its protection.