The thermal stability of thiol based DNA SAMs prepared on gold surfaces is an important parameter that is correlated to sensor lifetime. The thermal stability of DNA SAMs was evaluated in aqueous buffer through the use of fluorophore labeled DNA, a single crystal gold bead electrode, and microscopy. The stability of different crystallographic regions on the electrode was studied for thermal treatments up to 95 degrees C for 90 min. Using a in situ combinatorial surface analytical measurement showed that the crystallography of the underlying gold surface played a significant role, with the square or rectangular lattices (e.g., 110, 100, 210) having the highest stability. Surfaces with hexagonal lattices (e.g., 111, 311, 211) were less stable toward thermal treatments. These crystallographic trends were observed for both high and low coverage DNA SAMs. High coverage DNA SAMs were the most stable, with stability decreasing with decreasing coverage on average. Increasing DNA SAM coverage appears to slow the kinetics of thermal desorption, but the coordination to the underlying surface determined their relative stability. Preparing the DNA SAMs under nominally similar conditions were found to create surfaces that were similar at room temperature, but had significantly different thermal stability. Optimal DNA sensing with these surfaces most often requires low coverage DNA SAMs which results in poor thermal stability, which is predictive of a poor shelf life, making optimization of both parameters challenging. Furthermore, the crystallographically specific results should be taken into account when studying the typically used polycrystalline substrates since the underlying surface crystallography maybe different for different samples. It appears that preparing DNA SAMs with low coverage and significant thermal stability will be challenging using the current SAM preparation procedures.