Previous hypotheses of relationships within Dioon Lindl. indicated the presence of two large clades within the genus. However, relationships among species still remained unresolved. In this study, molecular phylogenetic analyses were performed with individual and combined data sets from the ITS region of the ribosomal DNA and the trnL–F region of the chloroplast DNA. To explore whether indels were a source of phylogenetically informative characters, indels were analyzed by excluding them from the analyses (Coding A); including them as ambiguous data (Coding B); as multistate (Coding C) and as binary characters (Coding D). We found that the rate of mutation in the ITS region is appropriate to solve most relationships at the species level. This is in contrast with the trnL–F rgion, which showed little variation. Our results show that most clades obtained during analyses correspond with previously recognized species within Dioon. This phylogeny groups the genus into two main clades that show clear biogeographic relationships between the species occurring along the Pacific Sierra Madre Occidental and the Atlantic Sierra Madre Oriental.