Atmospheric particulate matter can be strongly affected by smoke from biomass combustion, including wildfires, prescribed burns, and residential wood burning. Molecular source tracer techniques help determine contributions of biomass smoke to particle concentrations if representative source profiles are available. Various wood smoke source profiles have been generated for residential wood burning; however, few emission data are available for the combustion of biomass under open-burning conditions. Anhydrosugars, produced as thermal degradation products of cellulose and hemicellulose, are typically analyzed by gas chromatography-mass spectrometry (GC-MS) after chemical derivatization. A simpler alternative analytical method, based on high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), was developed here and utilized to measure several isomeric anhydrosugars (levoglucosan, mannosan, and galactosan) in primary smoke aerosol from various types of biomass and from different combustion conditions representative of prescribed and wildfires. Highly varying patterns were observed in the emission profiles of various molecular markers as a function of fuel type and combustion conditions. Emission factors of levoglucosan were a strong function of fuel type, combustion phase, and uphill versus downhill burn direction, varying from 36 to 1368 μg mg−1 organic carbon. Fuel type was the most important determinant, causing variations in emission factors of levoglucosan over an order of magnitude, while combustion phase and burn direction generally affected emission factors by a factor of 2–3. Mannosan and galactosan showed emission trends similar to levoglucosan. Levoglucosan emission factors from selected samples were compared to data obtained by two independent analytical methods, high-performance liquid chromatography (HPLC-MS) and GC-MS, showing rather good agreement. The HPAEC-PAD analytical method offers a simple alternative to GC-MS for future studies of aerosol concentrations of anhydrosugars, enabling more accurate estimates of contributions from biomass combustion to ambient particle concentrations.