BACKGROUND: Mitochondrial biogenesis is an essential process in all eukaryotes. Import of proteins from the cytosol into mitochondria is a key step in organelle biogenesis. Recent evidence suggests that a given mitochondrial protein does not take the same import route in all organisms, suggesting that pathways of mitochondrial protein import can be rewired through evolution. Examples of this process so far involve proteins destined to the mitochondrial intermembrane space (IMS). SCOPE OF REVIEW: Here we review the components, substrates and energy sources of the known mechanisms of protein import into the IMS. We discuss evolutionary rewiring of the IMS import routes, focusing on the example of the lactate utilisation enzyme cytochrome b2 (Cyb2) in the model yeast Saccharomyces cerevisiae and the human fungal pathogen Candida albicans. MAJOR CONCLUSIONS: There are multiple import pathways used for protein entry into the IMS and they form a network capable of importing a diverse range of substrates. These pathways have been rewired, possibly in response to environmental pressures, such as those found in the niches in the human body inhabited by C. albicans. GENERAL SIGNIFICANCE: We propose that evolutionary rewiring of mitochondrial import pathways can adjust the metabolic fitness of a given species to their environmental niche. This article is part of a Special Issue entitled Frontiers of Mitochondrial.