Volatile organic compounds (VOCs) of organisms provides a unique source for characterizing internal biological process and interactions that occur between organisms and their environment. Amphibians have unique chemodiverse profiles accounting for biogenic amines, peptides, proteins, alkaloids and more recently, VOCs discovered in some families. In spite the evidence that Dendrobatoidea superfamily has more than 500 alkaloids with promissory pharmaceutical applications, VOC surveys employing headspace (HS) methods have not been performed before in any of the species of the family. Therefore, the aim of this study is to determine the chemical diversity in the volatilomes of Colombian dendrobatids and its correlation with species differentiation and geographical separation. To accomplish this, comparisons between the VOC profiles collected with two sampling methods (HS-in vivo vs. HS-skin), of eight dendrobatid species (Allobates talamancae, Colostethus imbricolus, Dendrobates auratus, Dendrobates truncatus, Oophaga histrionica, Phyllobates aurotaenia, Phyllobates bicolor and Silverstoneia punctiventris), in four geographically localities separated (La Victoria, Tado, Santa Cecilia and Puerto Salgar) have been performed. We discovered that chemical profiles differed by extraction method used, species, and locality. Separately analyzing the results found with HS-in vivo and HS-skin, we have demonstrated that the combination of both metavariables species+locality explained the higher amount of variation on both datasets. However, as chemical profiles recovered by each method were different,similarities between VOC profiles of species and localities also differed. Taxonomic distances seemed to determine some of the similarity patterns, while others could be related more to geographic distances between localities. Annotation of the 30 main metabolites responsible for chemical differentiation, allowed to discover that HS methods successfully extracted amphibian alkaloids, besides other VOCs. The significance of the ecological functions that could be mediated by dendrobatid VOCs remained to be explored as well as the origin of these compounds.