Abstract A new species of Microsporidia, in the genus Thelohania, was found infecting fourth-instar larvae of Chironomus plumosus in Lake Winnebago, Wisconsin, causing 61% mortality over a 29-day period and inhibiting the pupation and emergence of adult flies. Larvae became infected before the final instar, but the large masses of spores were observed only in fourth-instar larvae in which the fat bodies were well developed. Fourth-instar larvae could not be infected in the laboratory, although 97% of first-instar larvae exposed to Thelohania sp. spores became infected. The incidence of parasitism of fourth-instar larvae in Lake Winnebago often varied from one area of the lake to another, and varied greatly from one generation to the next.