Technical Abstract: A striking characteristic of modern sugarcane is that all sugarcane cultivars (Saccharum spp.) share a common cytoplasm from S. officinarum. To explore the potential value of S. spontaneum cytoplasm, new Saccharum hybrids with a S. spontaneum cytoplasm were developed at the USDA-ARS, sugarcane research laboratory through a combination of conventional and molecular breeding approaches. In this study, we analyzed the genetic variability among the chloroplast genomes of four sugarcane cultivars, eight S. spontaneum clones, and three F1 progeny containing a S. spontaneum cytoplasm. Based on the complete chloroplast genome sequence information of two sugarcane cultivars (NCo 310 and SP 80-3280) and five related grass species (barley, maize, rice, sorghum, and wheat), 19 PCR primer pairs were designed targeting various chloroplast DNA (cpDNA) segments with a total length varying from 4,781 to 4,791 bp. Ten of the 19 cpDNA segments were polymorphic harboring 14 mutation sites [a 15-nt indel, a 5-nt indel, two poly (T), and 10 single nucleotide polymorphisms (SNPs)]. We demonstrate for the first time that the chloroplast genome of S. spontaneum was maternally inherited. Comparative sequence homology analyses clustered sugarcane cultivars into a distinctive group away from S. spontaneum and its progeny. Three mutation sites with consistent, yet species-specific, nucleotide composition were found, namely, an A/C transversion and two indels. The genetic variability among cpDNA of sugarcane cultivars and S. spontaneum will be useful information for the determination of maternal origin in Saccharum genus.