We herein morphologically diagnose the 5 natural groups of fish blood flukes and name them. Species of Chimaerohemecidae Yamaguti, 1971 infect chimeras, sharks, and rays (Chondrichthyes) and have C-shaped lateral tegumental spines and a non-sinusoidal testis or lack spines and have a sinusoidal testis. Species of Acipensericolidae n. fam. infect sturgeons and paddlefish (Acipenseriformes) and have a robust, bowl-shaped, pedunculate anterior sucker, lateral tegumental spines that are spike-like (not C shaped), an inverse U-shaped intestine (anterior ceca absent) with posterior ceca terminating near the excretory bladder, 6 testes (inter-cecal ovoid or oblong, lacking deep lobes; including 1 post-ovarian testis), a Laurer’s canal, and a dextral common genital pore. Species of Sanguinicolidae Poche, 1926 infect primarily later-branching freshwater ray-finned fishes (Teleostei) and have a diminutive anterior sucker, a medial esophageal swelling (pouch), short, radial ceca of approximately equal length or short anterior ceca plus an elongate, dendritic posterior cecum, testis with appendix-like lateral lobes, no Laurer’s canal, and separate or common genital pores. Species of Elopicolidae n. fam. infect ladyfishes, tarpons, and catadromous eels (Elopomorpha) and have a robust, bowl-shaped, pedunculate anterior sucker, lateral tegumental spines that are spike-like (can be lost in adult), short or indistinct anterior ceca, posterior ceca that terminate at level of the testis(es), a single testis or 2 testes, a Laurer’s canal present or absent, and a sinistral common genital pore and atrium. Species of Aporocotylidae Odhner, 1912 primarily infect later-branching marine and estuarine ray-finned fishes (Teleostei) and have a spheroid anterior sucker with concentric rows of circumferential spines or the spheroid anterior sucker is lost in adults or adults have a diminutive anterior sucker, a sinuous esophagus lacking a pouch, an X- or H-shaped intestine having 4 ceca, long anterior ceca (or secondarily lost), smooth posterior ceca that extend posteriad in parallel with respective body margin and terminate near the posterior body end, testis(es) that lack appendix-like lateral lobes, no Laurer’s canal, and a sinistral common genital pore or separate genital pores that are sinistral. Our 28S phylogeny recovered the fish blood flukes as monophyletic and each of the morphologically diagnosed families as monophyletic and sister to the remaining blood flukes infecting turtles and homeotherms. Acipensericolidae was recovered sister to the clade comprising Chimaerohemecidae + Sanguinicolidae and Elopicolidae + Aporocotylidae. The branching order and interrelationships of these families remains unsettled perhaps because of low taxon sampling among non-aporocotylids and extinction of intermediate taxa.