Native populations of lampreys are declining throughout the Great Lakes drainage basin due to habitat loss and degradation, anthropogenic stresses, and stream treatment with lampricides to control the exotic Petromyzon marinus (Sea Lamprey). We surveyed 19 streams across Pennsylvania to determine the presence/absence of six species that were historically found there. In 2011, we found four species—Lamptera aepyptera (Least Brook Lamprey), Lethenteron appendix (American Brook Lamprey), Ichthyomyzon greeleyi (Mountain Brook Lamprey), and Sea Lamprey—in 14 creeks statewide. In 2012, we found three species—Least Brook Lamprey, American Brook Lamprey, and Mountain Brook Lamprey— in 8 creeks in the Allegheny watershed. Lampreys appeared to be extirpated at some sites. Historically, Ichthyomyzon bdellium (Ohio Lamprey) and Ichthyomyzon fossor (Northern Brook Lamprey) were reported, but we did not observe these species during our study. Substrate analysis indicated ammocoetes preferred substrates with a particle diameter of <0.3 mm in shallow, warm water. In tributaries of Lake Erie, lampricide treatment to control Sea Lamprey may be a major reason for the population decline of native lamprey species.