Peroxisomes are dynamic organelles found in almost all eukaryotic cells and play a central role in intracellular metabolism. The number of peroxisomes is maintained through the balance of peroxisome biogenesis and degradation. Peroxisomes multiply by growth and division from preexisting peroxisomes but have also been shown to be synthesized de novo under experimental conditions. During de novo synthesis, pre-peroxisome vesicles mature in a stepwise process into functional peroxisomes. While the growth and division cycle is well studied, de novo synthesis, including whether it physiologically occurs, remains poorly understood. Although studies using several models have been proposed, the origin of the membranes required for peroxisome assembly remain controversial. This review provides an overview of the studies on de novo synthesis of peroxisomes in multiple organisms and discusses the evolutionary insights and biological meanings of peroxisome de novo synthesis.