Three species of freshwater sponges, Ephydatia muelleri (Lieberkühn, 1856), Ephydatia fluviatilis (Linnaeus, 1759), and Heteromeyenia baileyi (Bowerbank, 1863), were collected from western Pennsylvania streams and quarry lakes in the Ohio River watershed from 2014 to 2019. This is the first literature report of these sponge species in western Pennsylvania. Identification relied on spicule morphology and was supported by barcode analysis of partial mitochondrial DNA sequences from the cytochrome oxidase subunit 1 (COI) gene. These barcode sequences could differentiate Ephydatia from Heteromeyenia but could not reliably resolve species-level identifications due to low sequence variation. All three species were found in streams with little or no silt and situated downstream from lentic bodies of water. In addition, E. fluviatilis was found in quarry lakes on substrates of various pitches but not found on top of horizontal surfaces.