We investigated the potential of waste materials from wastewater treatment plants (WWTPs) to serve as an alternative lipid feedstock for biodiesel production. The average lipid recoveries from fat balls (46.4%) and primary scum (49.5-54.5%) were higher than the lipid recovery of primary sludge (15.8-16.4%). The yield of biodiesel produced from the extracted lipids ranged from 5.7 to 20.1%. There were considerable site- and season-dependent variations in the characteristics of the lipid waste materials. Radiocarbon analysis indicated the presence of fossil-derived carbon (26.0-42.0%) in the biodiesel obtained from wastewater lipids. Finally, we estimated the potential for biodiesel production from WWTP-derived lipids; about 333.0 metric tons of biodiesel per year could be produced from fat balls and primary scum in Japan. The results indicate that lipid-rich materials from WWTPs represent a valuable alternative feedstock for biodiesel production.