ABSTRACT: Among the species related to sugarcane, Saccharum spontaneum (L.) is a wild species with the greatest potential as a source of genetic variation to cope with biomass production in harsh environments. Due to its high yield, early vigor, ratooning ability, low input requirements and tolerance to various biotic and abiotic stresses, sugarcane breeders have shown interest in its contribution, as a donor of genes, to the development of high biomass energy canes. The conservation, evaluation and utilization of the genetic variability of S. spontaneum available in germplasm collections are critical for breeding, but, given the aggressive rhizomatous growth habit and the ability to propagate via seed dispersal, S. spontaneum is classified as a noxious weed in several nations, including the U.S.A. As a result, field trials are restrictive and few phenotypic analyses have been carried out on these collections. In the present study, a subset of 130 S. spontaneum accessions obtained from the World Collection of Sugarcane and Related Grasses in Miami, FL has been characterized phenotypically - with either morphological and agronomic traits (including composition analysis) or reaction to abiotic stress and genotypically - molecular markers (Simple Sequence Repeats - SSR). Using these data, a core collection has been established, genotypes with positive agronomic traits have been identified and are being used as parents for hybridization crosses, aimed at genetic improvement of sugarcane and energycane.