The production of fast pyrolysis bio-oil (FPBO) from secondary biomass is a promising technique for the production of liquid biofuels. However, the resulting FPBO is often viscous and chemically unstable, requiring significant upgrading while also being difficult to work with. To overcome this issue, FPBO can be hydrotreated using slurry hydrotreatment, which has a long history as a processing technique for upgrading viscous and difficult-to-work-with vacuum residues, and as such presents a solution. In this processing technique, the hydrotreatment catalyst is usually generated as dispersed MoS2 nanoparticles to improve feedstock-catalyst contact, as well as avert the diffusion problems that arise when utilizing a traditional, porous packed bed with a viscous feedstock. However, for such nanoparticulate dispersed catalysts, the nanoparticle structure and morphology play an important role in dictating activity and product distribution. Although much work has been done on characterizing dispersed MoS2 catalysts in the context of vacuum residues, such data is still scarce in the context of upgrading bio-oils. In this publication, we present a detailed characterization of in situ-generated MoS2 nanoparticles for the catalytic hydrotreatment of FPBO in a technology readiness level 5 (TRL5) slurry hydroprocessing plant. The results indicate a significant effect of the bio-based feedstock on the nanoparticle morphology, providing a starting point for further investigations into optimization of catalytic activity in the context of slurry hydrotrotreatment of bio-based feedstocks