Capsicum species produce fruits that synthesize and accumulate unique hot compounds known as capsaicinoids in placental tissues. The capsaicinoid biosynthetic pathway has been established, but the enzymes and genes participating in this process have not been extensively studied or characterized. Capsaicinoids are synthesized through the convergence of two biosynthetic pathways: the phenylpropanoid and the branched-chain fatty acid pathways, which provide the precursors phenylalanine, and valine or leucine, respectively. Capsaicinoid biosynthesis and accumulation is a genetically determined trait in chili pepper fruits as different cultivars or genotypes exhibit differences in pungency; furthermore, this characteristic is also developmentally and environmentally regulated. The establishment of cDNA libraries and comparative gene expression studies in pungent and non-pungent chili pepper fruits has identified candidate genes possibly involved in capsaicinoid biosynthesis. Genetic and molecular approaches have also contributed to the knowledge of this biosynthetic pathway; however, more studies are necessary for a better understanding of the regulatory process that accounts for different accumulation levels of capsaicinoids in chili pepper fruits.