Molecular analysis in plants requires the use of high quality DNA. Currently, there are a myriad of DNA extraction protocols, which still seems that their applicability across different species is not straightforward. The aim of the present study was to evaluate four different cetyl trimethylammonium bromide (CTAB)-based DNA extraction protocols for Heliconia species from lyophilized fully mature leaves. A split-plot design with two levels (six different species and four DNA extraction methods) was used, where treatments were distributed in a randomized block design with four replications. The response variables were total yield (µg) and purity (A260/A280 nm), and were determined by UVspectrophotometry and agarose gel electrophoresis, respectively. Additionally, EcoRI enzyme restriction digestion and amplification of the Hc_D6 locus marker were used to evaluate the functionality of the extracted DNA. Analysis of variance (ANOVA) results indicated that the extraction methods had a significant effect on the DNA yield (F = 8.51, df = 3, P<0.0001) and purity (F = 10.43, df = 3, P<0.0001). The best methods to obtain DNA from Heliconia spp. were those described by Michiels et al. (2003) and Sagahi-Maroof et al. (1984) with modifications.