Environmental contamination and regulation of longer-chain perfluoroalkyl substances (PFASs) such as perfluorooctane sulfonate (PFOS) has given rise to the increased use of shorter-chain PFASs as alternatives in new products, although confirmation of their presence in the environment remains limited. In this study, the PFAS alternative, perfluoro-1-butane-sulfonamide (FBSA) was identified for the first time in biota in homogenate samples of fish by liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-Q-ToF-MS) and quantified by ultra high pressure liquid chromatography -triple quadrupole mass spectrometry (UHPLC-QQQ-MS/MS). In one flounder (Platichthys flesus) muscle sample from the Western Scheldt, the Netherlands, FBSA was at 80.12 ng/g wet weight (w.w.) and exceeded only by PFOS. FBSA was also detected in 32 out of 33 samples of freshwater fish collected (2009-2010) from water bodies across Canada. In lake trout (Salvelinus namaycush) from northern Canada (e.g. Lake Kusawa (Yukon Territory), Great Bear Lake (Northwest Territories and in the Arctic) and Lake Athabasca (northern Alberta)) the concentration of FBSA ranged from below method detection limit (