The Cottonmouth, Agkistrodon piscivorus, is a semi-aquatic pitviper that occupies the southeastern U.S. west into Texas. Several previous studies have investigated the biogeographic history of A. piscivorus. It has been hypothesized that A. piscivorus was split into two separate populations during the last glacial maximum and climate change has impacted its distribution. Additionally, a geographically isolated population of A. piscivorus occurs at the western limit of the species' range in the Concho Valley of Texas. To investigate biogeography and population structure within A. piscivorus in Texas and throughout its range, we utilized amplified fragment length polymorphism (AFLP) and sequence data from cytochrome b (cyt-b). The AFLP data indicate a lack of gene flow between the population of A. piscivorus in the Concho Valley and other nearby populations. However, based on cyt-b, there is no genetic differentiation. The AFLP data for the entire species show a signature of two historic populations that have recently come into secondary contact. Finding two historic populations is consistent with previously published data based on mitochondrial DNA analyses; however, due to the rapid evolution rate of AFLP data, we were able to detect a high level of gene flow between these populations. We conclude that it is possible Texas and Florida served as refugia for A. piscivorus during the last glacial maximum, and, as the glaciers receded, the two populations expanded, coming into secondary contact. The subsequent gene flow has resulted in shared loci across the two populations. The difference between the conclusions drawn between our two markers and previous research is due to the different time scales that AFLP and cyt-b markers measure. The AFLP data provided a contemporary marker and cyt-b indicated historic separation.