The current work describes two new species of Gyrodactylus von Nordmann, 1832 collected from pipefish Syngnathus scovelli (Evermann et Kendall) and Syngnathus typhle L. during two separate gyrodactylosis episodes on fish held in a public aquarium located in northern Italy. The gyrodactylids collected from the skin, fins and gills of pipefish were subjected to a morphological analysis of the attachment hooks and the morphometric data were compared to the four species of Gyrodactylus previously described from syngnathid hosts, namely G. eyipayipi Vaughan, Christison, Hansen et Shinn, 2010, G. pisculentus Williams, Kritsky, Dunnigan, Lash et Klein, 2008, G. shorti Holliman, 1963 and G. syngnathi Appleby, 1996. Principal components analysis (PCA) of the morphological data indicated six clusters; two discrete groups among the specimens taken from the pipefish held in the Italian aquarium and four further groups representing G. eyipayipi, G. pisculentus, G. shorti and G. syngnathi. Molecular sequences of the ribosomal internal transcribed spacers (ITS1 and ITS2) and the 5.8S gene for the new species considered here were then compared with those available for other species in GenBank. The comparison did not reveal any identical match, supporting the morphological analysis that Gyrodactylus corleonis sp. n. from S. typhle and Gyrodactylus neretum sp. n. from S. scovelli represent distinct species. Both G. corleonis and G. neretum possess robust hamuli, marginal hook blades that curve smoothly from their sickle base to a point beyond the toe and, ventral bars with a broad median portion and a reduced membrane. Gyrodactylus corleonis, however, can be distinguished on the basis of its heart-shaped ventral bar; G. neretum has a 1:2 hamulus point:shaft ratio and a rectangular-shaped ventral bar. A redescription of the haptoral hard parts of the four species previously recorded on pipefish is also presented.