Members of Rhogeessa are hypothesized to have undergone speciation via chromosomal rearrangements in a model termed speciation by monobrachial centric fusions. Recently, mitochondrial cytochrome-b sequence data tentatively supported this hypothesis but could not explicitly test the model's expectations regarding interbreeding among karyotypic forms. These data showed potential evidence for hybridization or incomplete lineage sorting between the karyotypically distinct R. tumida and R. aeneus and identified multiple lineages of karyotypically identical R. tumida. Here, we present a more comprehensive test of speciation by monobrachial centric fusions in Rhogeessa. Our analysis is based on sequence data from two nuclear loci: paternally inherited ZFY and autosomal MPI genes. These data provide results consistent either with incomplete lineage sorting or ancient hybridization to explain alleles shared at low frequency between R. aeneus and R. tumida. Recent and ongoing hybridization between any species can be ruled out. These data confirm the presence of multiple lineages of the 2n=34 karyotypic form ("R. tumida") that are not each other's closest relatives. These results are generally consistent with speciation by monobrachial centric fusions, although additional modes of speciation have also occurred in Rhogeessa. Phylogeographic analyses indicate habitat differences may be responsible for isolation and divergence between different lineages currently referred to as R. tumida.