The molecular evaluation of morphologically based species delimitations of many herpetofauna has improved the understanding of evolutionary processes and the rigor of conservation efforts. Previous evidence for a deep lineage divide between south-eastern + northern subspecies of the softshell turtle Apalone spinifera (A. s. aspera + A. s. hartwegi + A. s. spinifera) and western subspecies (A. s. pallida + A. s. emoryi + A. s. guadalupensis) was re-examined with a broader sampling using mitochondrial and nuclear markers. The south-eastern + northern clade and the western clade maintained mitochondrial reciprocal monophyly. We molecularly confirmed a geographical boundary between these two clades in Oklahoma, and developed a phylogeographical hypothesis that invokes stream capture. We evaluated whether these mitochondrial lineages represent distinct species by surveying these clades for divergence at the nuclear intron R35 and two nuclear genes, Cmos and recombination activating gene 1 (RAG-1). The nuclear loci showed no phylogenetic resolution and only the nuclear intron exhibited significant nearest neighbour statistics. Taken together, the nuclear data suggest that taxonomic elevation of the two mitochondrial clades would be currently unjustified.