River cooters (Pseudemys concinna [Le Conte, 1830]) from the Pascagoula River (30°37′07.67″N; 88°36′44.53″W; Mississippi, U.S.A.) were infected by Spirorchis scripta Stunkard, 1923, and Spirorchis testiplexus n. sp. The new species resembles Spirorchis artericola (Ward, 1921) Stunkard, 1921, and Spirorchis innominatus Ward, 1921 (type species), by having a median esophageal diverticulum, ceca that extend slightly posterior to the genitalia, 10 testes, and a testicular column that extends anteriad to near the cecal bifurcation. Based on a comparative morphological study of existing type materials, vouchers, and published descriptions of similar congeners, S. testiplexus is unique by the combination of having a proportionally wider body, laterally directed cecal diverticula, deeply lobed testes, a testicular column that fills the intercecal space, and an external seminal vesicle that occupies the space between the posterior-most testis and the ovary and that passes dorsal or dorsolateral to the ovary. The large-subunit rDNA (28S) Bayesian inference phylogenetic analysis (including 16 turtle blood fluke species of 7 genera) showed a strongly supported monophyletic Spirorchis MacCallum, 1918, grouping sister to Spirhapalum siamensis Tkach, Snyder, and Vaughn, 2009.