Squash bugs cause widespread economic damage to cucurbit crops such as watermelon, cantaloupe, and squash and are vectors of Serratia marcescens, the bacterium causing cucurbit yellow vine disease. Trypanosome-like protozoans (TLP) were observed microscopically in bodies of adult, colony-reared (50% of bugs tested) and field-collected (38%) A. tristis. The goal of this study was to identify and characterize the TLP. Abdomen contents from both the squash bug colony and wild populations in and around Stillwater, OK were viewed at 100x and 1000x using dark-field microscopy. TLPs from colony insects were elongate and narrow (3 - 4µ by 0.75µ - 1µ) with a vertical groove and a single, polar flagellum (1.5 - 2µ). In contrast, those from wild bugs were rounded and tapered (1.7 - 4µ by 0.75 - 1µ) with short tip projections. These features match those of two different life stages of described trypanosomes. Samples from wild and colony-raised insects were subjected to PCR using primers Tr5S-L and Tr5S-R, which amplify a highly conserved 5S rRNA gene repeat region of trypanosomes. The PCR products, which were the expected 700bp in size, were sequenced and a BLAST search showed the closest match to be Blastocrithidia leptocoridis, family Trypanosomatidae. Matches of lower confidence included other trypanosomes Phytomonas, Leptomonas, Herpetemonas, and Crithidia. Our data confirm that the squash bug protozoan is a member of Trypanosomatidae. Future studies will address the potential role of this organism in squash bug biology and/or cucurbit disease.