Turtle acanthocephalans (genus Neoechinorhynchus) reside in the intestine of their definitive hosts and have been reported from a wide variety of turtle species. However, few reports exist of acanthocephalans in river cooters (Pseudemys concinna). We examined several eastern river cooters, P. c. concinna, from Arkansas and Oklahoma for acanthocephalans and generate the first sequence data for specimens of Neoechinorhynchus chrysemydis from this host. Molecular species identification was achieved by sequencing the complete internal transcribed spacer (ITS) gene and comparing it to ITS sequences for other species of Neoechinorhynchus from turtles and fishes available in GenBank. We also document a new state report for Neoechinorhynchus chrysemydis in Arkansas, as well as a new host record for N. chrysemydis in eastern river cooters in Oklahoma. Neoechinorhynchus chrysemydis appears to have relatively low host specificity among the species of turtle Neoechinorhynchus, and DNA sequencing can be a useful tool for investigating these host–parasite relationships.