Myxobolus diaphanus (Fantham, Porter et Richardson, 1940) was found in banded killifish Fundulus diaphanus (Lesueur) at several freshwater localities in Nova Scotia, including the type locality at the mouth of the Salmon River, Guysborough County. The new material, the first to be reported in 64 years, was used to supplement information on spore morphology, to document the site of development in the tissue, and to compare sequence data of the 18S rDNA to other studied myxobolids. Plasmodia with developed spores occurred in loose connective tissue of the head, the dermis (particularly in the roof of the mouth and at the base of fins), surface of the brain and ovary, muscle epimysium, and the submucosa of the intestine. Developed plasmodia containing spores were also found free in the lumen of the vena cava and within fluid-filled spaces of the skull, mandible and lower jaw. A phylogenetic analysis using 18S rDNA (878 bp) placed M. diaphanus in a terminal clade containing certain freshwater species of Henneguya, all of which occur in North America and have elongate spore bodies.