<p>The fish collection built up by Chiyomatsu Ishikawa (1860–1935), a Japanese zoologist who investigated the fish fauna of Lake Biwa in the 1890s, was reassessed for the first time in many years, and found to provide an important insight into the fish fauna of the lake at the end of the 1800’s. Now held in the National Museum of Nature and Science, Tsukuba, Japan, the collection includes 1,795 specimens (110 lots) collected from 12 sites in Lake Biwa and adjacent areas (i.e., Hikone, Imazu, Kaizu, Kaminyu, Lake Yogo, Maebara (= Maibara), Matsubara, Nagahama, Omi-Hachiman, Seta River, Shiotsu and Zeze). Thirty-three species, including 10 endemic species/subspecies, in 11 families were identified, including the type series of Pseudogobio zezera (=Biwia zezera). The majority of specimens were included in the following 7 species: Tanakia lanceolata, Acheilognathus tabira tabira, Candidia sieboldii, Ischikauia steenackeri, Sarcocheilichthys variegatus microoculus, Tachysurus nudiceps, and Plecoglossus altivelis altivelis (landlocked type). Such species are now seen infrequently along the lake foreshore (except for P. a. altivelis), the collection indicating a formerly rich fish fauna before the establishment of artificial influences (e.g., concrete revetments, invasion of alien species such as Micropterus nigricans and Lepomis macrochirus, and artificial control of water levels by the Seta River Weir). Although the occurrence of Acheilognathus longipinnis and Sarcocheilichthys variegatus variegatus in Lake Biwa has been unclear for a long time, examples of those species/subspecies in the collection provided evidence of their past distributions in the lake. Importantly, the collection sets a benchmark for any restoration of the past fish fauna of Lake Biwa.</p>