Abstract: Iron-sulfur clusters [Fe-S] are small, ubiquitous inorganic cofactors representing one of the earliest catalysts during biomolecule evolution and are involved in fundamental biological reactions, including regulation of enzyme activity, mitochondrial respiration, ribosome biogenesis, cofactor biogenesis, gene expression regulation, and nucleotide metabolism. Although simple in structure, [Fe-S] biogenesis requires complex protein machineries and pathways for assembly. [Fe-S] are assembled from cysteine-derived sulfur and iron onto scaffold proteins followed by transfer to recipient apoproteins. Several predominant iron-sulfur biogenesis systems have been identified, including nitrogen fixation (NIF), sulfur utilization factor (SUF), iron-sulfur cluster (ISC), and cytosolic iron-sulfur protein assembly (CIA), and many protein components have been identified and characterized. In eukaryotes ISC is mainly localized to mitochondria, cytosolic iron-sulfur protein assembly to the cytosol, whereas plant sulfur utilization factor is localized mainly to plastids. Because of this spatial separation, evidence suggests cross-talk mediated by organelle export machineries and dual targeting mechanisms. Although research efforts in understanding iron-sulfur biogenesis has been centered on bacteria, yeast, and plants, recent efforts have implicated inappropriate [Fe-S] biogenesis to underlie many human diseases. In this review we detail our current understanding of [Fe-S] biogenesis across species boundaries highlighting evolutionary conservation and divergence and assembling our knowledge into a cellular context.