Bio-oils produced from the rapid pyrolysis of poultry litter and hardwoods were observed to have considerable differences in their higher heating values, nitrogen contents, sulfur contents, and viscosities. In order to develop suitable applications of these potential biorefinery feedstocks, it is essential to characterized bio-oils with respect to their physico-chemical properties. Thus, bio-oils produced from the rapid pyrolysis of hardwood, broiler litter, and turkey litter were fractionated into acidic, basic, neutral and hydrocarbon fractions. The fractions were then characterized by gas chromatography, gas chromatography/mass spectrometry, thermogravimetric analysis, Fourier Transform infrared spectrometry, 13C-NMR, elemental analysis, and higher heating values (HHV). The bio-oils from the poultry litter had higher viscosities, higher nitrogen content, and higher HHVs than the corresponding samples from the hardwood. The hydrocarbon and neutral compounds content of the poultry litter oils were ten times higher than the corresponding hardwood biooils. Some oil fractions had very high levels of polymeric material and were very difficult to analyze by any of the above methods. Storage studies conducted on the oils showed that the viscosity increased very rapidly by probably two mechanisms; loss of low molecular weight volatile components, and polymerization of some fractions of the oil. There is a need to develop other characterization tools that will account for all fractions and analyze the bio-oils without necessarily fractionating them.