Tremendous plant metabolic diversity arises from phylogenetically restricted specialized metabolic pathways. Specialized metabolites are synthesized in dedicated cells or tissues, with pathway genes sometimes colocalizing in biosynthetic gene clusters (BGCs). However, the mechanisms by which spatial expression patterns arise and the role of BGCs in pathway evolution remain underappreciated. In this study, we investigated the mechanisms driving acylsugar evolution in the Solanaceae. Previously thought to be restricted to glandular trichomes, acylsugars were recently found in cultivated tomato roots. We demonstrated that acylsugars in cultivated tomato roots and trichomes have different sugar cores, identified root-enriched paralogs of trichome acylsugar pathway genes, and characterized a key paralog required for root acylsugar biosynthesis, SlASAT1-LIKE ( SlASAT1-L ), which is nested within a previously reported trichome acylsugar BGC. Last, we provided evidence that ASAT1-L arose through duplication of its paralog, ASAT1 , and was trichome-expressed before acquiring root-specific expression in the Solanum genus. Our results illuminate the genomic context and molecular mechanisms underpinning metabolic diversity in plants.