Identification of sugarcane hybrids is difficult when selections are based solely on morphological traits. Our objective was to combine morphological traits and molecular marker analysis to select F1 hybrids from two separate crosses between Djatiroto, a clone of Saccharum spontaneum, and elite sugarcane clones, LCP 85-384 (Cross 97-3144) and CP 62-258 (Cross 97-3146). The maternal inflorescences of Djatiroto were emasculated by submersion in a circulating 45°C hot-water tank for 10 min to minimize self-fertilization. Cross 97-3144 produced 4.7 g of seeds with 338 viable seeds per gram and Cross 97-3146 produced 2.4 g of seeds with 166 viable seeds per gram. After greenhouse germination, 96 progeny from each cross were evaluated in a field plot. Evaluations were conducted on the ratoon crops for stalk diameter (mm), juice Brix (percentage soluble solids), and a randomly amplified polymorphic DNA (RAPD) marker OPA-11-366 that was reproducibly amplified through PCR from the elite clones, but not the maternal S. spontaneum clone. Fifty progeny (52.1%) from Cross 97-3144 and 36 progeny (37.5%) from Cross 97-3146 inherited the RAPD marker. Five putative F1 progeny were selected from each cross, namely US 99-43, US 99-44, US 99-45, US 99-46 and US 99-47 from Cross 97-3144, and US 99-48, US 99-49, US 99-50, US 99-51 and US 99-52 from Cross 97-3146, based on their relatively larger stalk diameter, higher Brix and inheritance of the RAPD marker. The hybrid nature of these selected progeny was verified with sugarcane microsatellite markers. This is the first report of the development of Saccharum hybrids with the cytoplasm of S. spontaneum for breeding purpose through a combination of conventional and molecular breeding approaches. Availability of these F1 hybrids could enhance the genetic diversity of Saccharum germplasm and has enabled sugarcane geneticists and breeders to explore the possible contribution of S. spontaneum cytoplasm in the development of new sugarcane cultivars.