Congenital adrenal hyperplasia due to steroid 21-hydroxylase deficiency (21OHD) is the commonest inborn error in steroid biosynthesis. It is caused by mutations in the 21-hydroxylase gene (CYP21A2). A good genotypephenotype correlation exists allowing for prediction of the expressed adrenal phenotype. We performed functional and structural analysis of six novel CYP21A2 variants (p.Trp22Cys; p.Asp184Asn; p.Leu198Phe; p.Val305Gly; p.His310Asn; p.Thr443Asn), identified in one individual suspected to suffer from 21-hydroxylase deficiency, two men related to patients with 21-hydroxylase deficiency and in three individuals from the random sample of the general population from Northwest Spain. The novel variants were characterised in vitro using a yeast microsomal co-expression assay and a computational, three-dimensional CYP21A2 protein model. Kinetic assays constants were determined for the conversion of 17-hydroxyprogesterone and progesterone at 0.5, 1, 2 and 5 μM, respectively. p.Trp22Cys and p.Asp184Asn reduced wild-type activity to 7080%, p.Leu198Phe and p.Val305Gly showed enzyme activities similar to the wild-type. The variants p.His310Asn and p.Thr443Asn had 6570% residual activity. The in silico analysis was consistent with the in vitro findings. In vitro expression analysis revealed that p.Leu198Phe and p.Val305Gly are rare allelic variants not associated with a 21OHD phenotype. The activity of variants p.Trp22Cys and p.Asp184Asn are in a borderline area, which has been previously associated with non-classic CAH. Residual enzyme activities of p.His310Asn and p.Thr443Asn are compatible with non-classic 21OHD if homozygous or compound heterozygous. It is unlikely that either of the variants is associated severe classic CAH (salt wasting/simple virilising). Thus, our findings demonstrate the importance of CYP21A2 in vitro analyses in the molecular diagnosis of 21OHD to facilitate correct genetic counselling.