ABSTRACT Strigolactones, as signaling molecules and plant hormones, play essential roles in rhizosphere communication and plant growth and development. Strigolactones are structurally highly diversified, suggesting their biosynthesis is highly complex, with a range of different enzymes involved. Over the past decades, significant progress has been achieved in elucidating strigolactone biosynthesis in different plant species, which highlights particularly the importance of the cytochrome P450 family. Here, we give an overview of methodologies used for the discovery and functional characterization of the cytochrome P450s in strigolactone biosynthesis, classify and summarize these members discovered so far, review their biological importance, and discuss techniques in strigolactone biosynthesis research. Finally, we provide an outlook on some unsolved issues in strigolactone biology and discuss potential practical applications in agriculture.