Although bisexual reproduction has considerable evolutionary benefits, several all-female vertebrates exist. Unisexual salamanders in the genus Ambystoma are common around the Great Lakes region in eastern North America. They originated from a hybridization event that involved a female that shared a common ancestor with Ambystoma barbouri 2.4 to 3.9 million years ago but, unexpectedly, A. barbouri nuclear genomes were unknown in unisexuals. Unisexual salamanders steal sperm from donors of normally bisexual species, so their reproductive mode is described as kleptogenesis. Most known unisexuals are polyploid and they all possess at least one A. laterale genome. One or more other genomes are taken from sperm donors that may include A. jeffersonianum, A. laterale, A. texanum and A. tigrinum. We examined unisexual adults and larvae in a southern Ohio pond where unisexual individuals coexist with male A. barbouri. This population provided an opportunity to test hypotheses pertaining to the role of A. barbouri in the evolution of the disparate cytoplasmic and nuclear genomes in unisexual salamanders. Microsatellite DNA loci, mitochondrial DNA sequences and genomic in situ hybridization were used to identify the genomic constitution of individuals. A. barbouri was found to be an acceptable sperm donor for unisexuals but only contributed genomes in ploidy-elevated individuals. In the absence of A. jeffersonianum, this Ohio population is likely experiencing a recent switch in sperm donors from A. jeffersonianum to A. barbouri and demonstrates the evolutionary flexibility and dynamics of kleptogenesis.