Characterization of wild germplasm provides essential information on genetic diversity that breeders utilize for crop improvement. The potential of the sequence-related amplified polymorphism (SRAP) technique, which preferentially amplifies gene-rich regions, was evaluated to assess the genetic relationships among members of the Saccharum species. A panel of 31 SRAP primer combinations were used to score 30 genotypes of Saccharum officinarum, Saccharum spontaneum, Saccharum robustum, Saccharum sinense, Saccharum barberi and sugarcane hybrids, with Miscanthus and Erianthus included as outgroups. The amplifications produced 1364 DNA fragments for an average of 44 per primer combination, with 83% (1135) being polymorphic and 8.7% (119) being species specific. Based on the Dice index, all 30 genotypes were differentiated from each other with genetic similarity (GS) estimates ranging from 0.60 to 0.96 (mean = 0.79). Both the dendrogram [obtained by the unweighted pairgroup method with arithmetic mean or (UPGMA)] and the non-metric multidimensional scaling method grouped the genotypes according to their phylogenetic relationships. Erianthus and Miscanthus were separated as two outgroups (at GS levels of 0.56 and 0.72, respectively) into two major clusters: cluster I separated the S. robustum, S. sinense, S. barberi and hybrids as different subgroups with each one including some S. officinarum clones, while cluster II included the S. spontaneum clones, exclusively. A S. officinarum–S. spontaneum sequence comparison of some of the monomorphic and polymorphic bands revealed 65–90% homology with rice, corn or sugarcane sequences deposited in databases. The high discriminatory power coupled with the possibility that most of the amplicons may be amplifying gene-rich regions of the genome makes SRAP a potentially robust tool for genetic mapping aimed at marker-assisted introgression in sugarcane.