Multi-step biochemical pathways in plants is a challenging area of trait dissection as plants rapidly evolve novel specialized metabolites, often resulting in genus- or species-specific chemical compounds that are synthesized by unrelated gene families. Although the evolution of multi-step biochemical pathways catalyzed by proteins encoded by paralogous (duplicate) gene copies is understood to some extent, assembly of novel pathway steps across gene families is rarely investigated. Diterpenoids comprise one such area of divergent chemical traits in plants that are often restricted to limited taxa. As diterpenoid biosynthesis is catalyzed by famhilies of diterpene synthases and cytochrome P450s, natural experiments in biochemical pathway assembly have occurred repeatedly during evolution. The conifer Norway spruce (Picea abies) is an industrially and ecologically important tree in northern, central, and eastern Europe that produces a rich mixture of defensive diterpenoids in high abundance. Conifers, particularly the family Pinaceae, uniquely produce a mixture of C20 diterpene resin acids that can flush out and/or suffocate invading pests. As Norway spruce dominate large forests in Europe, and is used as an ornamental tree and for lumber, beetle invasions that decimate populations of Norway spruce are devastating. Due to the availability of molecular tools for genetic dissection and a published genome, this thesis attempts to answer questions about the evolution of novel diterpenoid pathways in Norway spruce using a combination of bioinformatics and biochemical analyses. Additionally, this work investigates regulation of terpene pathways and attempts to identify pathway steps that control pathway flux using transgenic manipulation of diterpene synthases and cytochrome P450s.