Polystomoides magdalenensis n. sp. is described from the buccal cavity of the Colombian slider Trachemys callirostris callirostris (Gray, 1856) in the middle of Magdalena River drainage, Colombia. The new species is characterized by a combination of traits, i.e., 29–35 genital spines, outer and inner hamuli length (123–150 and 54–63, respectively), testis size (261.4–554.5 × 388.2–768.4), marginal hook lengths (45–51), and pharynx length (150–333), as well as testis length/oral sucker length, testis length/pharynx length, and testis width/pharynx width ratios. This is the first species described from turtles in Colombia and the fifth reported in chelonians from South America.