Abstract Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders affecting key enzymes of cortisol biosynthesis. In the majority of cases the underlying cause are detrimental mutations in the steroidogenic cytochrome P450 enzyme 21-hydroxylase (CYP21A2). Early diagnosis via newborn screening programs in most Western countries and lifelong oral cortisol replacement therapy enable survival, however quality of life often is reduced and co-morbidities are substantially increased. Treatment is a major challenge as disease control can only be achieved with supraphysiological glucocorticoid doses. In addition, the currently available drugs cannot ideally mimic the circadian rhythm and stress adaption of cortisol secretion. Currently, disease severity is classified by residual enzyme activity. The goal of our research is to better understand the specific biophysico-chemical pathomechanism of 21-hydroxylase deficiency in order to enable causative therapeutic approaches. To this end, we investigated the structural and stability properties of six clinically relevant mutant variants of CYP21A2 (V282G/L, P31L, D323G, R484Q/W). Difficulty in purification of these CYP21A2 variants and various biophysical studies suggest that the proteins were less stable than wild-type (WT). Structural and thermal stability assessment by circular dichroism (CD) spectroscopy of recombinant, purified CYP21A2 mutant variants revealed high α-helical content for the WT (65% α-helix) and the mutants at the position 282 (V282G: 60.6 %, V282L: 57.6%). Other mutations (P31L, D323G, R484Q/W) disrupt the α-helical organization of CYP21A2 in exchange for a slight increase in ß-sheet content but mainly for random coil. Temperature dependent CD spectroscopy showed that all mutant variants have reduced thermal stability (Tm: 41.3 - 45,6°C) compared to the WT (Tm: 47.1°C). Tryptophane fluorescence showed that mutant variants of the protein were more prone to local unfolding at the hydrophobic core compared to WT using urea as denaturant. Furthermore, in UV/Vis spectroscopy at 280 nm and 418 nm we could demonstrate that all mutant variants had a reduced heme incorporation (A418/A280: 0.20 - 0.63) compared to WT (A418/A280: 0.88). Our results show that correct structural folding and stability pose a major problem in specific mutations involved in CAH. Therefore we propose that structural protein instability, play a key role in the pathophysiology of CAH and thus might constitute a novel tailored therapeutic target for the treatment of affected patients.