Introgression of novel cytoplasms into cotton (alloplasmic lines) via interspecific hybridization is being carried out to increase the genetic diversity of genotypes to potentially increase the genetic buffer against biotic and abiotic stresses. Following extensive backcrossing to a common genotype, all of the alloplasmic lines contain essentially the same nuclear DNA but each in a different cytoplasmic background (maternally inherited). Since nuclear DNA provides the primary basis of heredity, the alloplasmic lines rarely have notable morphological differences to distinguish them from cotton with normal cytoplasm. For those alloplasmic lines without distinguishing morphological features, seed contamination, outcrosses, etc. are difficult to detect and eliminate, especially in young seedlings. The advent of molecular markers has provided an opportunity to identify neutral variation sites in the sequence of the chloroplast and mitochondrial DNA of these lines. This study identifies a series of polymorphic markers that can be utilized to distinguish among maternal DNAs, i.e. cpDNA and mtDNA, associated with different Gossypium species cytoplasms. This provides a mechanism to assure that a purported alloplasmic line contains the alien cytoplasm, and to identify the species origin of the cytoplasm.