Pyrolysis bio-oils could be used in the future as biofuels or as a source of valuable oxygen-containing chemicals. To facilitate efficient exploitation of bio-oils, a detailed understanding of their structure is necessary. Over the past decade, petroleomic analysis has been widely applied to characterize pyrolysis bio-oils from the lignocellulosic biomass. Typically, a petroleomic analysis has been performed using high-resolution mass spectrometry (HRMS). HRMS has enabled the researchers to determine the molecular weights and molecular formulas of thousands of less volatile and nonvolatile, high-molecular-weight bio-oil compounds to obtain structural information that cannot be obtained using any other method. Here, we discuss the theoretical principles of HRMS and present an overview of the investigations regarding the petroleomic characterization of pyrolysis bio-oils and their key findings. In addition, this review outlines the current knowledge of the structure of bio-oil compounds detectable by HRMS. This could help us to understand the chemical composition of bio-oils in more detail and facilitate the design of processes for bio-oil upgrading and further utilization.