Recently, molecular techniques have gained the lead in the study of biodiversity, and have privileged genetic studies with fresh DNA samples from natural populations. Nevertheless, considering the great potential of information that lies in the biological collections, efforts have been made to obtain good quality DNA from these sometimes forgotten exemplars which have become an alternative for biodiversity studies in countries such as Colombia. Despite the literature reported difficulty in carrying out genetic investigations with museum exemplars due to the effect of fixing solutions on DNA integrity, in this study, DNA extracted from serpent tissues of the species Bothriechis schlegelii was evaluated in terms of its viability, of which some samples had more than 70 years of conservation in Colombian museums. Sixty samples from the biological collections of three museums were analyzed: the Serpentario y Coleccion Biologica de la Universidad del Cauca (Popayan), Museo de Historia Natural de la Universidad de Caldas (Manizales), and the Coleccion Biologica del Colegio San Jose del Instituto Tecnologico Metropolitano de Medellin. The DNA was extracted by two methods: a) Qiagen DNeasy Blood and Tissue commercial kit and b) phenol-chloroform-isoamyl alcohol. The DNA quality and quantity were evaluated, in both cases, by spectrophotometry and PCR amplification of the mitochondrial 12S, 16S, and COI genes and the ribosomal 18S and 28S genes. The best extraction results were obtained from the Qiagen DNeasy Blood and Tissue kit. The corresponding primers of the 18S and 28S genes showed the best amplification efficiency. It is concluded that organisms conserved in biological collections are material with high potential for use in molecular studies, independently of their preservation time.