The segregation of metabolic functions within discrete organelles is a hallmark of eukaryotic cells. These compartments allow for the concentration of related metabolic functions, the separation of competing metabolic functions, and the formation of unique chemical microenvironments. However, such organization is not spontaneous and requires an array of genes that are dedicated to the assembly and maintenance of these structures. In this review we focus on the genetics of peroxisome biogenesis and on how defects in this process cause human disease.