Bio-based polyols from different sustainable resources have been widely studied in recent years for applications in polyurethane (PU) production. The renewable materials have been proven as a viable alternative for petrochemical products. Tall oil-based polyols have a further advantage as they are produced from second-generation bio-based feedstock. Tall oil is a side-stream product of cellulose manufacture. The primary application of rigid PU foams is as a thermal insulation material in civil engineering and appliance industries. The properties of rigid PU foams can be tailored to suit various industrial applications by using polyols of different chemical structure. In this study, solely bio-based polyols were used to develop rigid PU foam formulations. PU polymer matrix with different crosslink density, i.e. molecular weight between the crosslinks, was developed by changing the ratio of tall oil-based polyols possessing high and low OH group functionality. The developed rigid PU foams had similar foaming kinetic characteristics, the apparent density of ~40 kg/m3, and the same closed cell content. A novel low global warming potential blowing agent Opteon 1100™ was used to achieve thermal conductivity as low as 18.6 mW/(m∙K). The thermal stability and compression strength properties were shown to increase with the growing crosslink density of the PU polymer matrix. Acknowledgements This research has been funded by the Latvian Council of Science, project Elimination of Rigid PU Foam Thermal Insulation Shortcomings by Determining and Adjusting the Factors Influencing Gas Permeability of Polymer Matrix (PURGE), project No. lzp-2019/1-0354