Thousand canker disease is a disease complex formed between susceptible walnut, butternut, and wingnut host trees, the fungal pathogen Geosmitha morbida, and the primary bark beetle vector Pityophthorus juglandis. Together, the plant pathogen and its principle vector induce cankers and excavate galleries beneath tree bark. TCD infestation and resulting damage cause tree decline, which when severe, can kill a tree in three to four years. Disease identification using traditional fungal isolation on agar cultures can take six weeks or more. A molecular protocol has been developed that can help identify G. morbida and P. juglandis within a day or two; however, the protocol requires an automated gel system that may be too expensive for most diagnostic laboratories. This research study is focused on enabling sample visualization using a conventional gel system rather than the more expensive equipment and better understanding the lower identification limits of an optimized DNA detection protocol. Optimized detection was assayed using both an automated system and conventional gel methodologies. Both systems provided a positive test result with as few as seven G. morbida hyphal cells or conidia. These findings enable an alternative detection approach that can replace the need for more expensive equipment. The resulting procedural improvements can be readily adopted by regulatory agents and diagnosticians charged with reducing the spread of thousand canker disease.