Intromugil alachuaensis n. sp. is described based on specimens collected from the flathead grey mullet (Mugil cephalus) from the Santa Fe River in Florida. The new species is the fourth recognized species in the genus and the second from North America, with the other 2 being confined to South America. Intromugil mugilicolus from Louisiana and Mississippi is redescribed based on the holotype and newly collected material that was not flattened prior to fixation. Two generic features not previously reported are apparent in the new material from I. mugilicolus and I. alachuaensis n. sp.: an armed oral sucker and a series of sacs containing glandular material arranged in symmetrical rows in the hermaphroditic duct. Intromugil alachuaensis differs from I. mugilicolus by having an oral sucker longer than wide, body spines smaller and lanceolate rather than longer and hastate, and smaller vitelline follicles. Intromugil alachuaensis n. sp. differs from Intromugil simonei by having a large elongated pharynx rather than a smaller subspherical one, a proportionately larger and longer oral sucker, and a longer prepharynx (greater than 1 pharyngeal length). Intromugil alachuaensis n. sp. differs from Intromugil annakohnae by having a longer than wide pharynx, a relatively large oral sucker, less extensive vitellarium, and smaller body spines. Comparison of more than 2,400 base-pair-long sequences of nuclear rDNA (partial 18S, complete ITS1, complete 5.8S, complete ITS2, and partial 28S) from I. mugilicolus and I. alachuaensis n. sp. reveals 110 pairwise differences, including gaps, thus supporting our proposal of a new species. These represent the first published sequences from species in this genus.