Acylsugars, specialized metabolites with defense activities, are secreted by trichomes of many solanaceous plants. Several acylsugar metabolic genes (AMGs) remain unknown. We previously reported multiple candidate AMGs. Here, using multiple approaches, we characterized additional AMGs. First, we identified differentially expressed genes between high- and low-acylsugar-producing F2 plants derived from a cross between Solanum lycopersicum and S. pennellii, which produce acylsugars ~1% and ~20% of leaf dry weight, respectively. Expression levels of many known and candidate AMGs positively correlated with acylsugar amounts in F2 individuals. Next, we identified lycopersicum-pennellii putative orthologs with higher nonsynonymous to synonymous substitutions. These analyses identified four candidate genes, three of which showed enriched expression in stem trichomes compared to underlying tissues (shaved stems). Virus-induced gene silencing confirmed two candidates, Sopen05g009610 [beta-ketoacyl-(acyl-carrier-protein) reductase; fatty acid synthase component] and Sopen07g006810 (Rubisco small subunit), as AMGs. Phylogenetic analysis indicated that Sopen05g009610 is distinct from specialized metabolic cytosolic reductases, but closely related to two capsaicinoid biosynthetic reductases, suggesting evolutionary relationship between acylsugar and capsaicinoid biosynthesis. Additionally, data mining revealed that orthologs of Sopen05g009610 are preferentially expressed in trichomes of several acylsugar-producing solanaceous species. Similarly, orthologs of Sopen07g006810 were identified as trichome-preferentially-expressed members, which form a phylogenetic clade distinct from those of mesophyll-expressed "regular" Rubisco small subunits. Furthermore, ?13C analyses indicated recycling of metabolic CO2 into acylsugars by Sopen07g006810 and shed light on how trichomes support high levels of specialized metabolite production. These findings have implications for genetic manipulation of trichome specialized metabolism in solanaceous crops, including tomato, potato, and tobacco.