Juvenile hormones (JHs) represent a family of sesquiterpenoid hormones in insects, and they play a key role in regulating development, metamorphosis, and reproduction. The last two steps of the JH biosynthetic pathway, epoxidation and methyl esterification of farnesoic acid to JH, are insect specific, and thus have long been considered a promising target for biorational insecticides. Recently, the enzymes involved in the last two steps have been molecularly identified: JH acid methyltransferase catalyzes the esterification step and the cytochrome P450 CYP15 enzyme catalyzes the epoxidation step. In this review, we describe the recent progress on the characterization of JH biosynthetic enzymes, with special focus on the function and diversity of the CYP15 family. CYP15 genes have evolved lineage-specific substrate specificity and regulatory mechanisms in insects, which appear to be associated with the lineage-specific acquisition of unique JH structure and function. In addition, the lack of CYP15 genes in crustacean (Daphnia pulex) and arachnid (Tetranychus urticae) species, whose genomes have been fully sequenced, may imply that CYP15 enzymes are an evolutionary innovation in insects to use the epoxide forms of methylated farnesoid molecules as their principal JHs. Molecular identification and characterization of CYP15 genes from broad taxa of insects have paved the way to the design of target-specific, biorational anti-JH agents.