A substantial opportunity exists to reduce carbon dioxide (CO2) emissions as well as dependence on foreign oil by developing strategies to cleanly and efficiently use biodiesel, a renewable, domestically available, alternative diesel fuel. However, biodiesel utilization presents several challenges, including decreased fuel energy density and increased emissions of smog-generating nitrogen oxides (NOx). These negative aspects can likely be mitigated via closed-loop combustion control provided the properties of the fuel blend can be estimated accurately, on-vehicle, in real-time. To this end, this paper presents a method to practically estimate the biodiesel content of fuel being used in a diesel engine during steady-state operation. The simple, generalizable, physically motivated estimation strategy presented utilizes information from a wideband oxygen sensor in the engine's exhaust stream, coupled with knowledge of the air-fuel ratio, to estimate the biodiesel content of the fuel. Experimental validation was performed on a 2007 Cummins 6.7 liter ISB series engine. Four fuel blends (0%, 20%, 50% and 100% biodiesel) were tested at a wide variety of torque-speed conditions. The estimation strategy correctly estimated the biodiesel content of the four fuel blends to within 4.2% of the true biodiesel content. Blends of 0%, 20%, 50% and 100% were estimated to be 2.5%, 17.1%, 54.2%, and 96.8% respectively. The results indicate that the estimation strategy presented is capable of accurately estimating the biodiesel content in a diesel engine during steady-state engine operation. This method offers a practical alternative to in-the-fuel type sensors, because wideband oxygen sensors are already in widespread production and are in place on some modern diesel vehicles today.