Context: CYP21A2 deficiency represents 95% of congenital adrenal hyperplasia cases (CAH), a group of genetic disorders that affect steroid biosynthesis. The genetic and functional analysis provides critical tools to elucidate complex CAH cases. One of the most accessible tools to infer the pathogenicity of new variants is in silico prediction. Objective: Analyze the performance of in silico prediction tools to categorize missense single nucleotide variants (SNVs) of the CYP21A2. Methods: SNVs of the CYP21A2 characterized in vitro by functional assays were selected to assess the performance of online single and meta predictors. SNVs were tested separately or in combination with the related phenotype (severe or mild CAH form). In total, 103 SNVs of the CYP21A2 (90 pathogenic and 13 neutral) were used to test the performance of 13 single-predictors and four meta-predictors. Results: SNVs associated with the severe phenotypes were well categorized by all tools, with an accuracy between 0.69 (PredictSNP2) and 0.97 (CADD), and Matthews' correlation coefficient (MCC) between 0.49 (PoredicSNP2) and 0.90 (CADD). However, SNVs related to the mild phenotype had more variation, with the accuracy between 0.47 (S3Ds&GO and MAPP) and 0.88 (CADD), and MCC between 0.18 (MAPP) and 0.71 (CADD). Conclusion: From our analysis, we identified four predictors of CYP21A2 pathogenicity with good performance. These results can be used for future analysis to infer the impact of uncharacterized SNVs' in CYP21A2.