ABSTRACT Spiny softshell turtles (Apalone spinifera) occur at the northwest limit of their range in Lake Champlain. This species, although widespread across North America, is listed as threatened in Vermont due to habitat destruction and disturbances of anthropogenic origin. The population of spiny softshell turtles in Lake Champlain is isolated from other North American populations and is considered as an independent management unit. Efforts to obtain information on the biology of spiny softshell turtles in Lake Champlain precede 1936 with conservation measures being initiated in 1987. Methods of studying spiny softshell turtles in Lake Champlain have included direct observation, mark-recapture, nest beach monitoring, winter diving, and radio telemetry. Each of these approaches has provided some information to the sum of what is known about A. spinifera in Lake Champlain. For example major nesting beaches, hibernacula, and home range size have been determined. Currently spiny softshell turtles primarily inhabit two areas within Lake Champlain, Missisquoi Bay and the mouth of the Lamoille River. However, the population structure and gene flow between spiny softshell turtles inhabiting the Lamoille and Missisquoi regions remained unknown. A GIS model was created and tested in order to identify additional nesting beaches used by spiny softshell turtles along the Vermont shores of Lake Champlain. Although some additional small potential nesting beaches were found, no additional major nesting sites were found. The GIS model identified the mouth of the Winooski River (the site of a historical population) as potentially suitable nesting habitat; however, no evidence of spiny softshell turtle nesting was found at this site. A series of methods developed for collecting molecular and population genetic data about spiny softshell turtles in Lake Champlain are described, including techniques for DNA extraction of various tissue types and the design of new primers for PCR amplification and sequencing of the mitochondrial control region (mtD-loop). Techniques for circumventing problems associated with DNA sequence alignment in regions of a variable numbers of tandem repeats (VNTRs) and the presence of heteroplasmy within some individuals are also described. The mtD-loop was found to be a suitable marker to assess the genetic structure of the Lake Champlain population of spiny softshell turtles. No significant genetic sub-structuring was found (FST=0.082, p=0.223) and an indirect estimate of the migration rate between Lamoille and Missisquoi regions of Lake Champlain was high (Nm>5.576). In addition to consideration of A. spinifera in Lake Champlain, the mtD-loop was modeled across 46 species in 14 families of extant turtles. The primary structure was obtained from DNA sequences accessed from GenBank and secondary structures of the mtD-loop were inferred, (from thermal stabilities) using the program Mfold, for each superfamiliy of turtles. Both primary and secondary structures were found to be highly variable across the order of turtles; however, the inclusion of an AT-rich fold (secondary structure) near the 3' terminus of the mtD-loop was common across all turtle families considered. The Cryptodira showed conservation in the primary structure at regular conserved sequence blocks (CSBs), but the Pluerodira displayed little conservation in the primary structure of the mtD-loop. Overall, greater conservation in secondary structure than primary structure was observed in turtle mtD-loop. The AT-rich secondary structural element near the 3' terminus of the mtD-loop may be conserved across turtles due to it serving a functional role during mtDNA transcription.