The phenotypic methods for identification of antifungal resistance are reliable procedures, and MIC determination by reference techniques is the gold standard to detect resistant clinical isolates. In recent years, progress has been made towards the description of resistance mechanisms at molecular level. There are methods of detection that can be useful for clinical laboratories, but lack of standardization precludes their full and effective integration in the routine daily practice. The molecular detection of Candida resistance to azoles and to echinocandins and of Aspergillus resistance to triazoles can be clinically relevant and could help to design more efficient prevention and control strategies. This text reviews the present state of the detection of mechanisms of resistance at the molecular level in Candida spp. and Aspergillus spp. and its relevance to clinical practice.