Tetraphyllidean cestodes of the genus Trilocularia have been reported from Squalus acanthias throughout this shark's global distribution, occupying both the stomach and spiral intestine. This study investigated whether variation seen in microthrix form among specimens within this host species is indicative of interspecific variation or is attributable to developmental transformation of a single cestode species. Sequence data for the 2 nuclear genes 28S rDNA (D1–D3) and ITS1, and the mitochondrial gene 16S rDNA, were generated for 22 specimens of Trilocularia; 3 from the stomach and 19 from the spiral intestine, including individuals exhibiting each of the 3 spinithrix forms found. Sequence data for 22 specimens were identical for the 2 nuclear genes. Some variation was seen in the mitochondrial gene but maximum likelihood analysis revealed no pattern with either site or microthrix type, suggesting microthrix variation may be developmental. To explore the developmental hypothesis further, a total of 118 juvenile worms from the stomach and 124 from the spiral intestine were collected from S. acanthias off the coast of Rhode Island and examined with either scanning electron microscopy (SEM) or light microscopy. SEM revealed differences in microthrix form between worms from the 2 sites. Of the stomach and spiral intestine worms examined, respectively, 98% versus 30% were scolopate, 2% versus 47% were aristate gladiate, and 0% versus 15% were lingulate in microthrix form. In addition, 3 of the 77 specimens from the spiral intestine examined with SEM exhibited mixtures of spinitriches. ANOVAs showed significant differences in the morphometrics of stomach worms, when compared to spiral intestine worms, as well as significat differences in the scolex width:below scolex width ratio of worms with scolopate spinitriches when compared to those with aristate gladiate or lingulate spinitriches. In combination, these data suggest that microthrix form may change over the course of development, indicating that caution should be exercised when interpreting microthrix patterns in the context of species designations.